Compositions comprising alpha-1C specific compounds

ABSTRACT

A method of treating benign prostatic hyperplasia in a subject which comprises administering to the subject a therapeutically effective amount of a compound which binds to a human α 1C  adrenergic receptor with a binding affinity greater than ten-fold higher than the binding affinity with which the compound binds to a human α 1A  adrenergic receptor, a human α 1B  adrenergic receptor, and a human histamine H 1  receptor, and, binds to a human α 2  adrenergic receptor with a binding affinity which is greater than ten-fold lower than the binding affinity with which the compound binds to such α 1C  adrenergic receptor. Compounds meeting these criteria are provided.

BACKGROUND OF THE INVENTION

[0001] Benign Prostatic Hyperplasia (BPH), also called Benign ProstaticHypertrophy, is a progressive condition which is characterized by anodular enlargement of prostatic tissue resulting in obstruction of theurethra. This results in increased frequency of urination, nocturia, apoor urine stream and hesitancy or delay in starting the urine flow.Chronic consequences of BPH can include hypertrophy of bladder smoothmuscle, a decompensated bladder and an increased incidence of urinarytract infection. The specific biochemical, histological andpharmacological properties of the prostate adenoma leading to thebladder outlet obstruction are not yet known. However, the developmentof BPH is considered to be an inescapable phenomenon for the aging malepopulation. BPH is observed in approximately 70% of males over the ageof 70. Currently, in the United States, the method of choice fortreating BPH is surgery (Lepor, H. Urol. Clinics North Amer., 17, 651(1990)). Over 400,000 prostatectomies are performed annually (data from1986). A medicinal alternative to surgery is clearly very desirable. Thelimitations of surgery for treating BPH include the morbidity rate of anoperative procedure in elderly men, persistence or recurrence ofobstructive and irritative symptoms, as well as the significant cost ofsurgery.

[0002] α-Adrenergic receptors are specific neurcreceptor proteinslocated in the peripheral and central nervous systems on tissuesthroughout the body. These receptors are important switches forcontrolling many physiological functions and, thus, represent importanttargets for drug development. in fact, many α-adrenergic drugs have beendeveloped over the past 40 years. Examples include clonidine,phenoxybenzamine and prazosin (treatment of hypertension), naphazoline(nasal decongestant), and apraclonidine (treating glaucoma).α-Adrenergic drugs can be broken down into two distinct classes:agonists (clonidine and naphazoline are agonists), which mimic thereceptor activation properties of the endogenous neurotransmitternorepinephrine, and antagonists (phenoxybenzamine and prazosin areantagonists), which act to block the effects of norepinephrine. Many ofthese drugs are effective but also produce unwanted side effects (forexample, clonidine produces dry mouth and sedation in addition to itsantihypertensive effects).

[0003] During the past 15 years a more precise understanding ofα-adrenergic receptors and their drugs has evolved through increasedscientific scrutiny. Prior to 1977, only one α-adrenergic receptor wasknown to exist. Between 1977 and 1988, it was accepted by the scientificcommunity that at least two α-adrenergic receptors—α₁ and α₂—existed inthe central and peripheral nervous systems. Since 1988, new techniquesin molecular biology have led to the identification of at least sixα-adrenergic receptors which exist throughout the central and peripheralnervous systems: α_(1A), α_(1B), α_(1C), α_(2A), α_(2B) and α_(2C)(Bylund, D. B., FASEB J., 6, 832 (1992)). It is not known preciselywhich physiological responses in the body are controlled by each ofthese receptors. In addition, many α-adrenergic drugs that weredeveloped before 1992 are not selective for any particular α-adrenergicreceptor. Many of these drugs produce untoward side effects which may beattributed to their poor α-adrenergic receptor selectivity.

[0004] Since the mid 1970's, nonselective α-antagonists have beenprescribed to treat BPH. In 1976, M. Caine, et al. (Brit. J. Urol., 48,255 (1976)), reported that the nonselective α-antagonistphenoxybenzamine was useful in relieving the symptoms of BPH. This drugmay produce its effects by interacting with α-receptors located on theprostate. However, this drug also produces significant side effectswhich severely limit its use in treating patients on a chronic basis.More recently, the α-adrenergic antagonists prazosin and terazosin havealso been found to be useful for treating BPH. However, these drugs alsoproduce untoward side effects. The most recently approved drug Proscar™(Merck) prescribed for BPH is not an α-adrenergic antagonist, but ratheracts by blocking 5-α-reductase. While Proscar is able to relievesymptoms, it is effective in only 30% of all patients, and requires aperiod of up to 6 months to show results.

[0005] From binding studies using cloned rat α_(1A), hamster α_(1B), andbovine α_(1C) receptors, and functional studies of antagonism in vitrousing human prostrate, I. Marshall, et al., concluded that the receptormediating contraction of the human prostrate is of the α_(1C) subtype(Marshall, I., et al., Brit. Pharmacol. Soc., (1992)).

[0006] Furthermore, using cloned human receptors the bindingcharacteristics of the known BPH drugs to various receptor subtypes havebeen determined, as described more fully hereinafter. Based upon suchbinding information and additional data, it has been observed that theside effects which occur with the drugs prazosin and terazosin may bedue to their poor selectivity for specific α-adrenergic receptors. Incontrast, indoramin is a drug which is slightly selective for the humanα_(1C) receptor relative to the other human α-adrenergic receptors, butit also interacts at human histamine H1 receptors. This compoundproduces untoward side effects which may be attributed to its activityat such H₁ receptors.

[0007] It would be desirable to provide methods and compounds whichallow the treatment of BPH but which avoid the production of sideeffects observed for all currently used medications.

[0008] From the binding information described hereinafter, it hasunexpectedly been discovered that compounds which are specific for anα_(1C) adrenergic receptor with a binding affinity greater than ten-foldhigher than the binding affinity with which the compounds bind to anα_(1A) adrenergic receptor, a human α_(1B) adrenergic receptor, and ahuman histamine H₁ receptor, and (b) bind to an α₂ adrenergic receptorwith a binding affinity which is greater than ten-fold lower than thebinding affinity with which the compounds bind to such α_(1C) adrenergicreceptor are effective for the treatment of BPH.

[0009] Furthermore, we have characterized several antagonists selectivefor the α_(1C) adrenergic receptor using a rat orthostatic hypotensionmodel to ascertain the vascular effects of drugs which may be indicativeof their ability to produce dizziness in patients, and observed thatwhile nonselective alpha 1 antagonists produce significant effects onorthostatic hypotension, selective alpha 1c antagonists do not producesignificant effects.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] A more complete understanding of the invention and many of itsadvantages will become apparent by reference to the detailed descriptionwhich follows when considered in conjunction with the accompanyingdrawings, wherein:

[0011]FIG. 1 illustrates compounds which are potent antagonists of thecloned human α_(1C) receptor.

[0012]FIG. 2 illustrates the correlation of inhibition constants(pK_(i)) for a series of α₁ antagonists at the cloned human α_(1A),α_(1B), and α_(1C) receptors with efficiency of blocking contraction ofhuman prostate tissue (pA₂).

SUMMARY OF HE INVENTION

[0013] The present invention provides a method of treating benignprostatic hyperplasia in a subject which comprises administering to thesubject a therapeutically effective amount of a compound which (a) bindsto a human α_(1C) adrenergic receptor with a binding affinity greaterthan ten-fold higher than the binding affinity with which the compoundbinds to a human α_(1A) adrenergic receptor, a human α_(1B) adrenergicreceptor, and a human histamine H₁ receptor, and (b) binds to a human α₂adrenergic receptor with a binding affinity which is greater thanten-fold lower than the binding affinity with which the compound bindsto such α_(1C) adrenergic receptor.

[0014] The present invention also provides a method of inhibitingcontraction of prostate tissue which comprises contacting the prostatetissue with an effective contraction-inhibiting amount of a compoundwhich (a) binds to a human α_(1C) adrenergic receptor with a bindingaffinity greater than ten-fold higher than the binding affinity withwhich the compound binds to a human α_(1A) adrenergic receptor, a humanα_(1B) adrenergic receptor, and a human histamine H₁ receptor, and (b)binds to a human α₂ adrenergic receptor with a binding affinity which isgreater than ten-fold lower than the binding affinity with which thecompound binds to such α_(1C) adrenergic receptor.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a method of treating benignprostatic hyperplasia in a subject which comprises administering to thesubject a therapeutically effective amount of a compound which (a) bindsto a human α_(1C) adrenergic receptor with a binding affinity greaterthan ten-fold higher than the binding affinity with which the compoundbinds to a human α_(1A) adrenergic receptor, a human α_(1B) adrenergicreceptor, and a human histamine H₁ receptor, and (b) binds to a human α₂adrenergic receptor with a binding affinity which is greater thanten-fold lower than the binding affinity with which the compound bindsto such α_(1C) adrenergic receptor.

[0016] Desirably, the compound used to practice the method of theinvention additionally binds to a calcium channel with a bindingaffinity which is greater than ten-fold lower than the binding affinitywith which the compound binds to the α_(1C) adrenergic receptor.

[0017] Alternatively or incrementally, the compound used to practice themethod of the invention also binds to a dopamine D₂ receptor with abinding affinity which is greater than ten-fold lower than the bindingaffinity with which the compound binds to the α_(1C) adrenergicreceptor.

[0018] Alternatively or incrementally, the compound used to practice themethod of the invention additionally binds to a dopamine D₂ receptorwith a binding affinity which is greater than ten-fold lower than thebinding affinity with which the compound binds to the α_(1C) adrenergicreceptor.

[0019] Alternatively or incrementally, the compound used to practice themethod of the invention additionally binds to any serotonin receptorwith a binding affinity which is greater than ten-fold lower than thebinding affinity with which the compound binds to the α_(1C) adrenergicreceptor.

[0020] Alternatively or incrementally, the compound used to practice themethod of the invention also binds to a human dopamine D₃ receptor witha binding affinity which is greater than ten-fold lower than the bindingaffinity with which the compound binds to the α_(1C) adrenergicreceptor.

[0021] Alternatively or incrementally, the compound used to practice themethod of the invention also binds to a human dopamine D₄ with a bindingaffinity which is greater than ten-fold lower than the binding affinitywith which the compound binds to the α_(1C) adrenergic receptor.

[0022] Alternatively or incrementally, the compound used to practice themethod of the invention also binds to a human dopamine D₅ receptor witha binding affinity which is greater than ten-fold lower than the bindingaffinity with which the compound binds to the α_(1C) adrenergicreceptor.

[0023] Alternatively or incrementally, the compound used to practice themethod of the invention also does not cause orthostatic fall in bloodpressure at a dosage effective to alleviate benign prostatichyperplasia.

[0024] Alternatively or incrementally, the compound used to practice themethod of the invention also does not cause orthostatic fall in bloodpressure in rats at a dosage 10 ug/kg.

[0025] A number of compounds have been identified or synthesized whichare useful in the practice of the invention. For example, the compoundhas the structure:

[0026] In another example, the compound has the structure:

[0027] In still another example, the compound has the structure:

[0028] In an additional example, the compound has the structure:

[0029] Included within the scope of the method of treating BPH in accordwith the invention are the use of both R and S enantiomers of thecompounds described which possess stereogenic centers, as well as theuse of pharmaceutically acceptable salts and complexes thereof.

[0030] The invention also provides a method of inhibiting contraction ofprostate tissue which comprises contacting the prostate tissue with aneffective contraction-inhibiting amount of a compound which (a) binds toa human α_(1C) adrenergic receptor with a binding affinity greater thanten-fold higher than the binding affinity with which the compound bindsto a human α_(1A) adrenergic receptor, a human α_(1B) adrenergicreceptor, and a human histamine H₁ receptor, and (b) binds to a human α₂adrenergic receptor with a binding affinity which is greater thanten-fold lower than the binding affinity with which the compound bindsto such α_(1C) adrenergic receptor.

[0031] The activity of compounds at the different human receptors wasdetermined in vitro using cultured cell lines that selectively expressthe receptor of interest. These cell lines were prepared by transfectingthe cloned cDNA or cloned genomic DNA or constructs containing bothgenomic DNA and cDNA encoding the human α-adrenergic, serotonin,histamine, and dopamine receptors as further described in detail inExample 9 hereinbelow.

[0032] In connection with this invention, a number of cloned humanreceptors discussed herein, either as plasmids or as stably transfectedcell lines, have been made pursuant to, and in satisfaction of, theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure, and are made withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852. Specifically, these deposits have been accorded ATCCAccession Numbers as follows: Designation ATCC Accession No. DateL-α_(1A) CRL 11138 Sep. 25, 1992 L-α_(1B) CRL 11139 Sep. 25, 1992L-α_(1C) CRL 11140 Sep. 25, 1992 L-α_(2A) CRL 11180 Nov. 6, 1992L-NGC-α_(2B) CRL 10275 Oct. 25, 1989 L-α_(2C) CRL 11181 Nov. 6, 1992pcEXV-H₁ 75346 Nov. 6, 1992 pcEXV-H₂ 75345 Nov. 6, 1992 pcEXV-D₂ 75344Nov. 6, 1992

[0033] The data shown in the accompanying Tables 1 and 2 indicate thatthe α_(1C)-specific receptor antagonists which satisfy the criteria asdefined herein have significant efficacy in the inhibition ofcontraction of human prostate tissue. This in vitro property isrecognized in the art as correlating with efficacy in treating benignprostatic hyperplasia in vivo.

[0034] The present invention therefore provides a method of treatingbenign prostatic hyperplasia, which comprises administering a quantityof any of the α_(1C) receptor antagonists defined as herein in aquantity effective against BPH. The drug may be administered to apatient afflicted with benign prostatic hyperplasia by any conventionalroute of administration, including, but not limited to, intravenous,intramuscular, oral, subcutaneous, intratumoral, intradermal, andparenteral. The quantity effective against BPH is between 0.001 mg and10.0 mg per kg of subject body weight.

[0035] The method of treating BPH disclosed in the present invention mayalso be carried out using a pharmaceutical composition comprising any ofthe α_(1C) receptor antagonists as defined herein and a pharmaceuticallyacceptable carrier. The composition may contain between 0.05 mg and 500mg of an α_(1C) receptor antagonist, and may be constituted into anyform suitable for the mode of administration selected. Compositionssuitable for oral administration include solid forms, such as pills,capsules, granules, tablets, and powders, and liquid forms, such assolutions, syrups, elixers, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

[0036] The drug may otherwise be prepared as a sterile solid compositionwhich may be dissolved or suspended at the time of administration usingsterile water, saline, or other appropriate sterile injectable medium.Carriers are intended to include necessary and inert binders, suspendingagents, lubricants, flavorants, sweeteners, preservatives, dyes, andcoatings.

[0037] Optimal dosages to be administered may be readily determined bythose skilled in the art, and will vary with the particular α_(1C)receptor antagonist in use, the strength of the preparation, the mode ofadministration, and the advancement of the disease condition. Additionalfactors depending on the particular patient being treated will result ina need to adjust dosages, including patient age, weight, diet, and timeof administration.

[0038] The following Experimental Details are set forth to aid in anunderstanding of the invention, and are not intended, and should not beconstrued, to limit in any way the invention set forth in the claimswhich follow thereafter.

[0039] Experimental Details.

[0040] Prazosin, 5-methylurapidil, and S-niguldipine were obtained fromResearch Biochemicals, Inc. A30360(4-fluoro-4-(8-fluoro-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)butyrophenonehydrochloride) was obtained from Aldrich Chemical Co. Other compoundswere prepared according to the examples which follow.

EXAMPLE 1 Synthesis of Terazosin Hydrochloride N-(2-Furoyl)piperazine

[0041] This compound and its preparation has been described in GreatBritain Patents 1,390,014 and 1,390,015. Piperazine hexahydrate (194 g,1 mole) was dissolved in 250 ml H₂O. The solution was acidified to pH4.5 with 6 N HCl. Furoyl chloride (130.5 g, 1 mole, Aldrich) was addedalong with 10% NaOH solution at such a rate that the pH was maintainedat 4.5. After 1 hour, the solution was made basic (pH=8.5) with NaOHsolution. The reaction mixture was continuously extracted withchloroform for 36 hours. The CHCl₃ extract was dried over MgSO₄, andfiltered. Distillation gave 108.2 g product (60%), b.p. 132°-138° C./0.6mm Hg, m.p. 69°-70° C.

N-(Tetrahydro-2-furoyl)piperazine

[0042] The furoylpiperazine of Example 1 was converted to thehydrobromide salt (m.p. 173°-175° C.). This salt (39.0 g) in 250 mlmethyl alcohol and 9.0 g Raney nickel was hydrogenated at 3 atm. Afteruptake of H₂ ceased, the catalyst was filtered, the solventconcentrated, and the residue crystallized from isopropyl alcohol togive 35.2 g. tetrahydrofuroylpiperazine HBr, m.p. 152°-156° C. This wassuspended in 20 ml H₂O. Then 10.5 g 50%, NaOH solution was added slowlyfollowed by 2.0 g solid Na₂CO₃. This was extracted with 4×100 mlportions of warm CHCl₃. The CHCl₃ extractions were distilled to give22.5 g tetrahydrofurolylpiperazine, b.p. 120°-125° C./0.2 mm Hg.

2[4-(Tetrahydro-2-furoyl)piperazinyl]-4-amino-6,7-dimethoxyquinazolinehydrochloride

[0043] To 7.00 g 2-chloro-4-amino-6,7-dimethoxyquinazoline (LancasterSynthesis) in 50 ml methoxyethanol was added 10.8 g,tetrahydrofurolylpiperazine, and the mixture refluxed 3 hours. The clearsolution was concentrated and an aqueous solution of potassiumbicarbonate was added. The resultant solid that formed was filtered andwashed with water. It was then added to methanol and the resultingsuspension was acidified with a solution of hydrogen chloride inisopropyl alcohol. The resulting solution was concentrated and theresidue crystallized from isopropyl alcohol giving 8.12 g. of product,m.p. 278°-279° C.

EXAMPLE 2 Preparation of Indoramin4-Benzamido-1-[2-(3-indolyl)ethylpyridinium Bromide

[0044] A solution of 4-benzamidopyridine (1.98 g) and3-(2-bromoethyl)indole (2.24 g) in EtOH (15 ml) was refluxed for 2hours, and the crystallized product (3.13 g, mp 264-266° C.) wascollected by filtration from the hot reaction mixture. Recrystallizationgave the hydrate.

3-[2-4-Benzamidopiperid-1-yl)ethyl]indole (Indoramin)

[0045] 4-Benzamido-1-[2-(3-indolyl)ethyl]pyridinium bromide (3.0 g) in91% EtOH (300 ml) containing Et₃N (0.8 g) was hydrogenated in thepresence of freshly prepared W-7 Raney Ni catalyst (ca. 3 g) at 28.12kg/cm² and 50° for 4 hours. After filtering off the catalyst, thefiltrate was evaporated and the residue was shaken with CHCl₃ and 2 NNaOH. The resulting insoluble material (1.61 g, mp 203-206° C.) wascollected and dried. Recrystallization from EtOH gave the product (1.34g), as colorless needles.

EXAMPLE 3 Preparation of 1-(3-benzoylpropyl)-4-benzamidopiperidine

[0046] A mixture of 4-chlorobutyrophenone (447 mg, 2.45 mmol),4-benzamidopiperidine (500 mg, 2.45 mmol) and K₂CO₃ (338 mg, 2.45 mmol)was heated up in boiling water bath for 1 hour. The reaction mixture wasportioned between water and CHCl₃. The organic layer was separated anddried over Na₂SO₄. After filtration and removal of solvent, the residuewas purified by chromatography (SiO₂, MeOH:CHCl₃, 5:95).Recrystallization from AcOEt/hexane gave a white powder (78 mg, 8.2%).mp 143-144° C.; ¹H NMR (CD₃OD, 400 MHz) δ1.65 (dq, J₁=3.16 Hz, J₂=11.9Hz, 2H), 1.90-2.00 (m, 4H), 2.18 (t, J=11.9 Hz, 2H), 2.48 (m, 2H),3.00-3.10 (m, 4H), 3.88 (m, 1H), 7.40-8.00 (m, 10H); Mass spectrum(M+1)⁺ at m/z 351.

EXAMPLE 4 Preparation of1-[3-(4-chlorobenzoyl)propyl]-4-benzamidopiperidine

[0047] A mixture of 3-(4-chlorobenzol)propyl bromide (640 mg, 2.45mmol), 4-benzamidopiperidine (500 mg, 2.45 mmol) and K₂CO₃ (1.01 g, 7.34mmol) in 50 ml of acetone was heated up to refluxing condition for 48hours. The solid was removed by filtration. Concentration of filtrate invacuo gave a yellowish solid, which was purified by chromatography(SiO₂, MeOH:CHCl₃, 5:95). 320 mg (33.9%) of white powder was obtained ¹HNMR (CDCl₃, 300 mHz) δ1.46 (dq, J₁=1.0 Hz, J₂=8.4 Hz, 2H), 1.90-2.10 (m,4H), 2.16 (m, 2H), 2.43 (t, J=6.9 Hz, 2H), 2.80-2.90 (m, 2H), 2.97 (t,J=6.9 Hz, 2H), 3.97 (m, 1H), 5.92 (d, J=7.8 Hz, 1H, N-H), 7.40-8.00 (m,9H); Product was converted to HCl salt and recrystallized withMeOH/Et₂O, mp 243-244° C.; Calcd for C₂₂H₂₅ClN₂O₂.HCl.H₂O: C, 60.15, H,6.37, N, 6.37; Found: C, 60.18, H, 6.34, N, 6.29.

EXAMPLE 5 Preparation of SKF-104856 1-[(4-Chlorophenyl)thio}-2-propanone

[0048] Chloroacetone (32.3 g, 0.347 mol) was added to a mixture of4-chlorothiophenol (50 g, 0.347 mmol) and sodium hydroxide (14 g, 0.347mol) in water (400 ml) and the mixture was stirred at 25° C. for 1 hour.The mixture was extracted with ethyl ether and the organic phase waswashed with water, dried with magnesium sulfate and concentrated to give69 g (99%) of 1-[(4-chlorophenyl)thio]-2-propanone.

5-Chloro-3-methylbenzo(b)thiophene

[0049] 1-[(4-Chlorophenyl)thio}-2-propanone (50 g, 0.25 mol) was addedto polyphosphoric acid (300 g) and the mixture was stirred as thetemperature was gradually raised to 120° C. as an exotherm started. Themixture was stirred at 130° C. for 1 hour, diluted with water, extractedwith ethyl ether and the organic phase was dried and concentrated. Theresidue was stirred in methanol (200 ml), filtered and the filtrateconcentrated to give 17.5 g (40%) of 5-chloro-3-methylbenzo(b)thiophene:bp 120° C. (0.6 mm Hg).

Ethyl5-chloro-3-methylbenzo(b)thiophene-2-carboxylate

[0050] n-Butyllithium in hexane (2.6 M, 2.3 ml) was added to a solutionof 5-chloro-3-methylbenzo(b)thiophene (1,0 g, 6 mmol) in ethyl ether (20ml) stirred at 0° C. under argon. The mixture was stirred for 30 minutesand transferred slowly under argon pressure to a stirred solution ofethyl chloroformate (0.63 g, 6 mmol) in ethyl ether (20 ml). The mixturewas stirred at 0° C. for 30 minutes and at 25° C. for 1.5 hours. Themixture was treated with water and the organic phase was dried,concentrated and triturated with hexane to give 1.0 g (67%) of ethyl5-chloro-3-methylbenzo(b)thiophene-2-carboxylate: mp 92.5-94 ° C.

Ethyl 3-bromomethyl-5-chlorobenzo(b)thiophene-2-carboxylate

[0051] A mixture of ethyl5-chloro-3-methylbenzo(b)thiophene-2-carboxylate (9.0 g, 0.035 mol),N-bromosuccinimide (6.53 g, 0.037 mol) and benzoyl peroxide (130 mg) incarbon tetrachloride (150 ml) was refluxed and illuminated with sunlampfor 2 hours. The resulting suspension was cooled, filtered and thefilter cake was triturated with methanol to give 9.9 g, (85%) of themethanol-insoluble ethyl3-bromomethyl-5-chlorobenzo(b)thiophene-2-carboxylate: mp 148-150° C.

Ethyl5-Chloro-3-[N-(2,2-dimethoxyethyl)-N-methyl(aminomethyl)]benzol(b)thiophene-2-carboxylate

[0052] A mixture of ethyl3-bromomethyl-5-chlorobenzo(b)thiophene-2-carboxylate (11 g, 0.033 mol),methylaminoacetaldehyde dimethyl acetal (4.76 g, 0.04 mol) and potassiumcarbonate (11.4 g, 0.8 mol) in dry acetone (200 ml) was stirred for 48hours, filtered and the filtrate concentrated to give 11.8 g, (96%) ofethyl5-chloro-3-(N-2,2-dimethoxyethyl)-N-methyl(aminomethyl)benzol(b)thiophene-2-carboxylate.

Ethyl7-chloro-3,4-dihydro-4-methylthieno[4,3,2-ef]-[3]benzazepine-2-carboxylate

[0053] Ethyl5-chloro-3-[N-(2,2-dimethoxyethyl)-N-methyl(aminomethyl)]benzo[b]thiophene-2-carboxylate(3.0 g, 8.1 mmol) was added in portions to trifluoromethanesulfonic acid(10 ml) stirred at 0° C. under argon. The mixture was stirred at 25° C.for 45 minutes and diluted with water. The mixture was basified withaqueous sodium hydroxide and extracted with ethyl ether to give ethyl7-chloro-3,4-dihydro-4-methylthieno-[4,3,2-ef][3]benzazepine-2-carboxylate.

Ethyl7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-carboxylate

[0054] Diborane in tetrahydrofuran (1 M, 40 ml) was added to a solutionof ethyl7-chloro-3,4-dihydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-carboxylate(2.8 g) in tetrahydrofuran (30 ml) stirred at 0° C. The mixture wasrefluxed for 3 hours and stirred at 25° C. for 18 hours, cooled, treatedwith methanol (50 ml), refluxed for 18 hours and concentrated. Theresidue was triturated with ethyl ether-hexane (3:1) to give 1.6 g (84%)of ethyl7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-carboxylate:mp138-140° C. The free base was treated with hydrogen chloride to giveethyl7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-carboxylatehydrochloride: mp 240° C.

7-Chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-methanol

[0055] A solution of ethyl7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4.3.2-ef][3]benzazepine-2-carboxylate(4.0 g, 12.9 mmol), in ethyl ether (48 ml) was treated with lithiumaluminum hydride (0.53 g, 14 mmol). The mixture was stirred for 1.5hours, cooled and treated carefully with water (2.0 ml), 10% sodiumhydroxide (1.0 ml) and water (2.0 ml). The resulting mixture wasfiltered and the solvent evaporated to give 1.9 g (57%) of7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-methanol:mp 184-185° C.

7-Chloro-3, 4,5,6-tetrahydro-4-methylthieno-4,3,2-ef][3]benzazepine-2-carboxaldehyde

[0056] A solution of7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepine-2-methanol(1.6 g, 6 mmol) in dichloromethane (150 ml) was stirred under argon withactivated manganese dioxide (8.3 g) for 2 hours. The mixture wasfiltered through Celite™ and the filtrate was dried with magnesiumsulfate and concentrated to give a 63% yield of7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef[[3]benzazepine-2-carboxaldehyde.

7-Chloro-2-ethenyl-3,4,5,6-tetrahdyro-4-methylthieno[4,3,2-ef][3]benzazepine(SKF-104856)

[0057] Sodium hydride (60% dispersion in mineral oil. 3.8 mmol) wasadded to a stirred solution of methyltriphenylphosphonium bromide (1.35g, 3.8 mmol) in dry tetrahydrofuran (30 ml) and stirred for 15 minutes.The mixture was treated with a solution of7-chloro-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]-benzazepine-2-carboxaldehyde,prepared as in Example 3, (0.5 g, 1.9 mmol) in dimethylformamide (4 ml),stirred at 25° C. for 16 hours, quenched with ice and extracted withethyl acetate. The organic phase was washed, dried and concentrated andthe residue was chromatographed on silica gel eluted with a gradient ofmethylene chloride to methanol-methylene chloride (3.5:96.5). Theproduct was treated with hydrogen chloride to give 0.2 g (35%) of7-chloro-2-ethenyl-3,4,5,6-tetrahydro-4-methylthieno[4,3,2-ef][3]benzazepinehydrochloride: mp 234-236° C.

EXAMPLE 6 2-Hydroxymethyl-1,2,3,4-tetrahydronaphthalene

[0058] A solution of 1,2,34-tetrahydro-2-naphthoic acid (2.50 g, 14.2mmol) in 100 ml THF was treated with LiAlH₄ (681 mg, 17.04 mmol) and thereaction mixture was heated at reflux for 5 hours. The suspension wascooled to 0° C. and quenched by addition of solid Na₂SO₄▪10H₂O. Themixture was stirred at room temperature for 4 hours. The solid wasremoved by filtration. Concentration of filtrate in vacuo gave ayellowish oil (2.28 g, 98.8%) ; ¹H NMR (CDCl₃, 300 MHz) δ1.43 (m, 1H),2.00 (m, 2H) 2.51 (dd, J₁=16.5 Hz, J₂=10.8 Hz, 1H) , 2.85 (m, 3H), 3.65(dd, J₁=6.3 Hz, J₂=1.2 Hz, 2H), 7.09 (s, 4H).

2-Bromomethyl-1,2,3,4-tetrahydronaphthalene

[0059] A solution of 2-hydroxymethyl-1,2,3,4-tetrahydronaphthalene (2.28g, 14.0 mmol) in 100 ml of CH₂Cl₂ was treated with PBr₃ (1.28 g, 4.73mmol) at 0° C. The mixture was stirred at room temperature for 72 hoursthen poured onto 100 g of ice. The organic layer was isolated, washedwith 10% K₂CO₄ aqueous solution, H₂O, sat'd brine, and then dried overNa₂SO₄. After filtration and removal of solvent, the residue waspurified by chromatography (SiO₂, EtOAc:hexane, 1:10) to give acolorless oil (1.33 g, 41.6%); ¹H NMR (CDCl₃, 300 MHz) δ1.55 (m, 1H),2.11 (m, 1H), 2.11 (m, 2H), 2.58 (dd, J₁=16.2 Hz, J₂=10.2 Hz, 1H),2.80-3.10 (m, 3H), 3.45 (d, J=6.3 Hz, 2H), 7.10 (m, 4H).

2-[(4-Methoxyphenethyl)aminomethyl]-1,2,3,4-tetrahydronaphthalene(Compound 11)

[0060] A solution of 2-bromomethyl-1,2,3,4-tetrahydronaphthalene (1.33g, 5.91 mmol) and 4-methoxyphenethylamine (1.79 g, 11.8 mmol) in 50 mlof EtOH was refluxed for 48 hours. After removal of EtOH in vacuo, theresidue was dissolved in 100 ml of CHCl₃, washed with 10% K₂CO₃, H₂O,sat'd brine, and then dried over Na₂SO₄. Filtration followed byevaporation of solvent gave a yellow oil, which was purified bychromatography (SiO₂, MeOH:CHCl₃, 5:95) to a give a yellowish oil (1.03g, 58.9%). The product was converted to HCl salt, crystallization withMeOH/Et₂O gave a white powder. mp 274-275° C.; Calcd for C₂₀H₂₅NO.HCl:C, 72.37, H, 7.91, N, 4.22; Found C, 72.40, H, 7.76, N, 4.13.

EXAMPLE 7 4,4-Diphenylpiperidine hydrochloride

[0061] A mixture of 4-piperidone monohydrate hydrochloride (15.0 g, 97.6mmol, 1.00 equiv, Aldrich) and AlCl₃ (130 g, 976 mmol, 10.0 equiv) inanhydrous benzene (600 mL) was stirred at reflux for 4 hours. Ice (300g) and water (50 mL) were added, the mixture was filtered, and the solidwas washed with toluene and dried to afford 19.2 g (72%) of off-whitesolid, which was pure by ¹H NMR. Recrystallization from ethanol gave theanalytically pure sample: m.p. 300-301° C.; ¹H NMR (300 MHz, CD₃OD)δ2.65 (m, 4 H), 3.18 (m, 4 H), 7.18 (m, 2 H), 7.30 (m, 8 H); Anal.Calcd. for C₁₇H₁₉N. HCl: C, 74.57; H. 7.36; N, 5.12. Found: C, 74.32; H,7.34; N, 5.02. The free base was generated by addition of the above saltto dilute aqueous sodium hydroxide and extraction with CH₂Cl₂. Theorganic phase was dried over MgSO₄ and concentrated to give a lightbrown solid: IR (neat) 2942.8, 1494.5, 1445.9 cm⁻¹; CIMS (NH₃) m/e 238(M+1)⁺.

3-(4,4-Diphenylpiperidin-1-yl)propionitrile

[0062] To a suspension of 4,4-diphenylpiperidine hydrochloride (195 mg,0.712 mmol, 1.0 equiv) in ETOh (1.5 mL) was added triethylamine (0.25mL, 1.83 mmol, 2.6 equiv) followed by acrylonitrile (0.13 mL, 2.01 mmol,2.8 equiv). The resulting solution was stirred at room temperature underargon for 15 minutes and then concentrated. Water was added, and themixture was extracted three times with EtOAc. The combined organicextracts were dried over MgSO₄ and concentrated to give 170 mg (87%) oftan solid, which was used for the next reaction without purification.m.p. 95-96° C.; ¹H NMR (300 MHz, CDCl₃) δ2.37 (m, 2H), 2,46 (m, 4H),2.52 (m, 6H), 7.12 (m, 2H), 7.23 (m, 8H); ¹³C NMR (75 MHz, CDCl₃)δ16.65, 36.71. 45.08, 50.78, 54.13, 119.70, 126.48, 127.78, 129.11,147.87; IR (neat) 2944.4, 2821.0, 1495.5, 1445.9 cm⁻¹.

1-(3-Aminopropyl)-4,4-diphenylpiperidine

[0063] To a stirred solution of3-(4,4-diphenylpiperidine-1-yl)propionitrile (2.00 g, 6.89 mmol, 1.0equiv) in anhydrous THF (20 mL) under argon was added a solution of BH₃in THF (1.0 M, 24.1 mL, 24 mmol, 3.5 equiv) at room temperature. Themixture was refluxed for 4.5 hours and then cooled to room temperature.Aqueous HCl (6 N, 50 mL) was added and stirring was continued for 1hour. The mixture was basified to pH 9 by addition of 6 N aq. NaOH,extracted 3 times with CH₂Cl₂, dried over MgSO₄ and concentrated. Theresidue was purified by flash chromatography (SiO₂, EtOAc-MeOH, 9:1,followed by EtOAc-MeOH-isopropylamine (60:10:1), followed byEtOAc-MeOH-isopropylamine (40:10:2) to give 1.35 g (66%) of tan solid:m.p. 98-99° C.; ¹H NMR (300 MHz, CDCl₃) δ1.64 (tt, J=7.7 Hz, 2H), 2.33(br t, J=7.2 Hz, 2H), 2.50 (m, 8H), 2.76 (br t, J=6.5 Hz, 2H), 3.06 (brs, 2H), 7.13 (m, 2H), 7.26 (m, 8H); ¹³C NMR (75 MHz, CDCl₃) δ29.79,36.80, 41.41, 45.24, 51.25, 57.41, 126.30, 127.77, 128.97, 148.11; IR(neat) 3361.5 cm⁻¹; CIMS (NH₃) m/e 295 (M+1)⁺.

Acetoacetic acid N-[3-(4,4-diphenylpiperidin-1-yl)propyl]amide

[0064] Diketene (0.44 mL, 5.68 mmol, 1.3 equiv, Aldrich) was added atroom temperature to a stirred solution of1-(3-aminopropyl)-4-,4-diphenylpiperidine (1.288 g, 4.37 mmol, 1.0equiv) in anhydrous toluene (15 mL) under argon, and stirring wascontinued for 48 hours. The mixture was concentrated to give 1.294 g(78%) of white solid, which was used for the next reaction withoutpurification: ¹H NMR (300 MHz, CDCl₃) δ1.70 (tt, J=6.4, 6.4 Hz, 2H),2.23 (s, 3H), 2.44 (br t, J=6.5 Hz), 2.49-2.67 (m, 8H) , 3.32 (br t,J=5.8 Hz), 3.36 (s, 2H), 7.16 (m, 2H), 7.27 (m, 8H).

2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acidN-(3-(4,4-diphenylpiperidine-1-yl)propyl]amide methyl ester

[0065] A solution of acetoacetic acidN-[3-(4,4-diphenylpiperidin-1-yl)propyl]amide (365 mg, 0.964 mmol, 1.0equiv), methyl 3-aminocrotonate (138 mg, 1.20 mmol, 1.2 equiv, Aldrich),and 4-nitrobenzaldehyde (181 mg, 1.20 mmol, 1.2 equiv, Aldrich) inisopropanol was refluxed under argon for 60 hours. The mixture wascooled to room temperature and concentrated, and the residue was dilutedwith CH₂Cl₂, washed with water, dried over MgSO₄, and concentrated. Theresidue was purified by flash chromatography (SiO₂, EtOAc, followed byEtOAc-MeOH, 19:1 and 9:1) to give 147.8 mg (25%) of yellow solid: ¹H NMR(300 MHz, CDCl₃) δ1.55 (m, 2H), 2.14 (s, 3H) , 2.15-2.50 (m, 10H) , 2.32(s, 3H) , 3.20 (m, 1H), 3.37 (m, 1H), 3.54 (s, 3H), 5.00 (s, 3H), 5.48(br s), 6.98 (br t, J=4.9 Hz, 1H), 7.14-7.30 (m, 10H), 7.39 (dm, J=8.7Hz, 2H), 8.05 (dm, J=8.7 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃) δ18.74, 20.64,25.61, 36.77, 40.20, 42.26, 45.03, 51.16, 51.61, 58.08, 100.65, 109.71,124.35, 126.46, 127.61, 128.84, 129.06, 135.52, 146.96, 147.10, 154.55,168.22, 168.70; IR (neat) 1680, 1610, 1515, 1340 cm⁻¹; MS (FAB) m/e 609(M+H)⁺.

2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acidN-[3-(4,4-diphenylpiperidin-1-yl)-propyl]amide methyl esterhydrochloride hydrate (Compound 2)

[0066] To a solution of2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydro-pyridine-3,5-dicarboxylicacid N-[3-(4,4-diphenylpiperidin-1-yl)propyl]amide methyl ester (147.8mg, 0.243 mmol, 1.0 equiv) in EtOH (2 mL) was added a solution of HCl inether (1.0 M, 0.24 mL, 0.24 mmol, 1.0 equiv). Addition of ethyl acetate(3 mL) followed by heating gave a clear solution. Slow cooling of thissolution, followed by filtration gave 91 mg of yellow crystalline solid:m.p. 182-183° C.; Anal. Calcd. for C₃₆H₄₀N₄O₅.HCI.H₂O: C, 65.20, H,6.54; N, 8.45. Found: C, 65.30; H, 6.28; N, 8.15.

EXAMPLE 8 3-(4,4-Diphenylpiperid-1-yl)-propanol

[0067] 4,4-Diphenylpiperidine (40 g), 3-bromopropanol (24.7 g, Aldrich),powdered potassium carbonate (116.4 g) and approximately 1 g ofpotassium iodide (in 500 ml of a 1:1 mixture of dioxane and 1-butanol)were heated for about 48 hours under reflux and with vigorous stirring.After cooling, the mixture was filtered, and the filtrate wasconcentrated. The oily residue was taken up in ethyl acetate, and thesolution was filtered again. Concentrating the filtrate to drynessyielded the product in the form of a yellowish, oily residue whichslowly solidifies to a wax-like product (yield: 44.8 g). Hydrochloricacid in ether produced the hydrochloride (m.p.: 226° to 227° C.), whichwas recrystallized from 2-propanol.

Acetoacetic acid 3-(4,4-diphenylpiperidin-1-yl)propyl ester

[0068] 23.6 g of 3-(4,4-diphenylpiperid-1-yl)-propanol were dissolved in100 ml of absolute toluene, and 16 ml of a 50% strength solution ofdiketene in acetone were added with stirring. After standing for severaldays at room temperature (monitored by thin layer chromatography), themixture was concentrated, and the residue was dried under high vacuum.The pale yellow, viscous oil which remains was employed without furtherpurification for the next stage.

2,6-Dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxy-ylicacid [3-(4,4-diphenylpiperidin-1-yl)propyl] ester methyl ester

[0069] A solution of methyl 3-aminocrotonate (265 mg, 2.3 mmol, 1.0equiv), 4-nitrobenzaldehyde (348 mg, 2.3 mmol, 1.0 equiv), andacetoacetic acid 3-[4,4-diphenylpiperidin-1-yl)propyl] ester (872 mg,2.3 mmol, 1.0 equiv) in isopropanol was refluxed under argon withstirring for 68 hours. Cooling and removal of solvent gave a residue,which was purified by flash chromatography (SiO₂, EtOAc-hexane, 1:1 and1:2, followed by EtOAc) to afford 717 mg (51%) of yellow solid: ¹H NMR(300 MHz, CDCl₃) δ1.73 (m, 2H), 2.22 (m, 2H), 2.30-2.51 (m, 8H) , 2.34(s, 3H) , 2.35 (s, 3H), 3.63 (s, 3H), 4.05 (dt, J=2.1, 7.9 Hz, 2H), 5.06(s, 1H), 5.73 (br s, 1H), 7.14 (m, 2H), 7.27 (m, 8H), 7.42 (dm, J=8.8Hz, 2H) , 8.06 (dm, J=8.8 Hz, 2H) ; ¹³C NMR (75 MHz, CDCl₃) δ15.30,19.65, 26.32, 36.11, 39.88, 44.60, 50.60, 51.12, 55.34, 62.66, 102.99,107.55, 123.39, 125.67, 127.12, 128.33, 128.65, 144.80, 144.93, 146.36,147.50, 154.78, 166.91, 167.43; IR (neat) 1698.0, 1684.7, 1517.5, 1345.7cm⁻¹; CIMS (NH₃) 610 (M+1)⁺, 553, 338.

2,6-Dimethyl-4-(4-nitrophenyl)-1 4-dihydropyridine-3,5-dicarboxylic acid[3-(4,4-diphenylpiperidin-1-yl)propyl] ester methyl ester hydrochloride(Compound 8)

[0070] To a solution of2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydro-pyridine-3,5-dicarboxylicacid [3-(4,4-diphenylpiperidine-1-yl)-propyl] ester methyl ester (710mg, 1.16 mmol, 1.0 equiv) in EtOH (5 mL) was added a solution of HCl inether (1.0 M, 1.5 mL, 1.5 mmol, 1.3 equiv). The solvents were removedand the residue was dissolved in CH₂Cl₂. This solution was addeddropwise to 25 mL of ether to afford, after filtration, 500 mg of yellowcrystalline solid: m.p. 152-153° C. Anal. Calcd. for C₃₆H₃₉N₃O₆.HCl: C,66.92; H, 6.24; N, 6.50. Found: C, 66.70; H, 5.99; N, 6.27

EXAMPLE 9 Protocol for the Determination of the Potency of α₁Antagonists

[0071] The activity of compounds at the different human receptors wasdetermined in vitro using cultured cell lines that selectively expressthe receptor of interest. These cell lines were prepared by transfectingthe cloned cDNA or cloned genomic DNA or constructs containing bothgenomic DNA and cDNA encoding the human α-adrenergic, serotonin,histamine, and dopamine receptors as follows:

[0072] α_(1A) Human Adrenergic Receptor: The entire coding region of α1A(1719 bp) (Sequence I.D. No. 1), including 150 basepairs of 5′untranslated sequence (5′ UT) and 300 bp of 3′ untranslated sequence (3′UT), was cloned into the BamHI and ClaI sites of the polylinker-modifiedeukaryotic expression vector pCEXV-3, called EXJ.HR. The constructinvolved the ligation of partial overlapping human lymphocyte genomicand hippocampal cDNA clones: 5′ sequence were contained on a 1.2 kbSmaI-XhoI genomic fragment (the vector-derived BamHI site was used forsubcloning instead of the internal insert-derived SmaI site) and 3′sequences were contained on an 1.3 kb XhoI-ClaI cDNA fragment (the ClaIsite was from the vector polylinker). Stable cell lines were obtained bycotransfection with the plasmid α1A/EXJ (expression vector containingthe α1A receptor gene) and the plasmid pGCcos3neo (plasmid containingthe aminoglycoside transferase gene) into LM(tk⁻), CHO, and NIH3T3cells, using calcium phosphate technique. The cells were grown, in acontrolled environment (37° C., 5% CO₂), as monolayers in Dulbecco'smodified Eagle's Medium (GIBCO, Grand Island, N.Y.) containing 25 mMglucose and supplemented with 10% bovine calf serum, 100 units/mlpenicillin g, and 100 μg/ml streptomycin sulfate. Stable clones werethen selected for resistance to the antibiotic G-418 (1 mg/ml), andmembranes were harvested and assayed for their ability to bind[³H]prazosin as described below (see “Radioligand Binding assays”).

[0073] α_(1B) Human Adrenergic Receptor: The entire coding region of α1B(1563 bp) (Sequence I.D. No. 3), including 200 basepairs and 5′untranslated sequence (5′ UT) and 600 bp of 3′ untranslated sequence (3′UT), was cloned into the EcoRI site of pCEXV-3 eukaryotic expressionvector. The construct involved ligating the full-length containing EcoRIbrainstem cDNA fragment from λ ZapII into the expression vector. Stablecell lines were selected as described above.

[0074] Human α_(1C) Adrenergic Receptor: The entire coding region of α1C(1401 bp) (Sequence I.D. No. 5), including 400 basepairs of 5′untranslated sequence (5′ UT) and 200 bp of 3′ untranslated sequence (3′UT), was cloned into the KpnI site of the polylinker-modifiedpCEXV-3-derived eukaryotic expression vector, EXJ.RH. The constructinvolved ligating three partial overlapping fragments: a 5′ 0.6 kbHincII genomic clone, a central 1.8 EcoRI hippocampal cDNA clone, and a3′ 0.6 Kb PstI genomic clone. The hippocampal cDNA fragment overlapswith the 5′ and 3′ genomic clones so that the HincII and PstI sites atthe 5′ and 3′ ends of the cDNA clone, respectively, were utilized forligation. This full-length clone was cloned into the KpnI site of theexpression vector, using the 5′ and 3′ KpnI sites of the fragment,derived from vector (i.e., pBluescript) and 3′-untranslated sequences,respectively. Stable cell lines were selected as described above.

[0075] Radioligand Binding Assays: Transfected cells from culture flaskswere scraped into 5 ml of 5 mM Tris-HCl, 5 mM EDTA, pH 7.5, and lysed bysonication. The cell lysates were centrifuged at 1000 rpm for 5 min at4° C., and the supernatant was centrifuged at 30,000×g for 20 min at 4°C. The pellet was suspended in 50 mM Tris-HCl, 1 mM MgCl₂, and 0.1%ascorbic acid at pH 7.5. Binding of the α1 antagonist [³H]prazosin (0.5nM, specific activity 76.2 Ci/mmol) to membrane preparations of LM(tk−)cells was done in a final volume of 0.25 ml and incubated at 37° C. for20 min. Nonspecific binding was determined in the presence of 10 μMphentolamine. The reaction was stopped by filtration through GF/Bfilters using a cell harvester. Inhibition experiments, routinelyconsisting of 7 concentrations of the tested compounds, were analyzedusing a non-linear regression curve-fitting computer program to obtainKi values.

[0076] α₂ Human Adrenergic Receptors: To determine the potency of α₁antagonists at the α₂ receptors, LM(tk−) cell lines stably transfectedwith the genes encoding the α_(2A), α_(2B), and α_(2C) receptors wereused. The cell line expressing the α_(2A) receptor is designatedL-α_(2A), and was deposited on Nov. 6, 1992 under ATCC Accession No. CRL11180. The cell line expressing the α_(2B) receptor is designatedL-NGC-α_(2B), and was deposited on Oct. 25, 1989 under ATCC AccessionNo. CRL10275. The cell line expressing the α_(2C) receptor is designatedL-α_(2C), and was deposited on Nov. 6, 1992 under ATCC Accession No.CRL-11181. Cell lysates were prepared as described above (seeRadioligand Binding Assays), and suspended in 25 mM glycylglycine buffer(pH 7.6 at room temperature). Equilibrium competition binding assay wereperformed using [3H]rauwolscine (0.5 nM), and nonspecific binding wasdetermined by incubation with 10 μM phentolamine. The bound radioligandwas separated by filtration through GF/B filters using a cell harvester.

[0077] Human Histamine H₁ Receptor: The coding sequence of the humanhistamine H₁ receptor, homologous to the bovine H₁ receptor, wasobtained from a human hippocampal cDNA library, and was cloned into theeukaryotic expression vector pCEXV-3. The plasmid DNA for the H₁receptor is designated pcEXV-H1, and was deposited on Nov. 6, 1992 underATCC Accession No. 75346. This construct was transfected into COS-7cells by the DEAE-dextran method. Cells were harvested after 72 hoursand lysed by sonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The celllysates were centrifuged at 1000 rpm for 5 min at 4° C., and thesupernatant was centrifuged at 30,000×g for 20 min. at 4° C. The pelletwas suspended in 37.8 mM NaHPO₄, 12.2 mM KH₂PO₄, pH 7.5. The binding ofthe histamine H₁ antagonist [³H]mepyramine (1 nM, specific activity:24.8 Ci/mM) was done in a final volume of 0.25 ml and incubated at roomtemperature for 60 min. Nonspecific binding was determined in thepresence of 10 μM mepyramine. The bound radioligand was separated byfiltration through GF/B filters using a cell harvester.

[0078] Human Histamine H₂ Receptor: The coding sequence of the human H₂receptor was obtained from a human placenta genomic library, and clonedinto the cloning site of PCEXV-3 eukaryotic expression vector. Theplasmid DNA for the H₂ receptor is designated pcEXV-H2, and wasdeposited on Nov. 6, 1992 under ATCC Accession No. 75346. This constructwas transfected into COS-7 cells by the DEAE-dextran method. Cells wereharvested after 72 hours and lysed by sonication in 5 mM Tris-HCl, 5 mMEDTA, pH 7.5. The cell lysates were centrifuged at 1000 rpm for 5 min at4° C., and the supernatant was centrifuged at 30,000×g for 20 min at 4°C. The pellet was suspended in 37.8 mM NaHPO₄, 12.2 mM K2PO₄, pH 7.5.The binding of the histamine H₂ antagonist [³H]tiotidine (5 nM, specificactivity: 70 Ci/mM) was done in a final volume of 0.25 ml and incubatedat room temperature for 60 min. Nonspecific binding was determined inthe presence of 10 μM histamine. The bound radioligand was separated byfiltration through GF/B filters using a cell harvester.

[0079] Human Serotonin Receptors:

[0080] 5HT_(1Dα), 5HT_(1Dβ), 5HT_(1E), 5HT_(1F) Receptors: The celllysates of LM(tk−) clonal cell line stably transfected with the genesencoding each of these 5HT receptor-subtypes were prepared as describedabove. The cell line for the 5HT_(1Dα) receptor, designated asLtk-8-30-84, was deposited on Apr. 17, 1990, and accorded ATCC AccessionNo. CRL 10421. The cell for the 5HT_(1Dβ) receptor, designated asLtk-11, was deposited on Apr. 17, 1990, and accorded ATCC Accession No.CRL 10422. The cell line for the 5HT_(1E) receptor, designated 5HT_(1E)-7, was deposited on Nov. 6, 1991, and accorded ATCC AccessionNo. CRL 10913. The cell line for the 5HT_(1F) receptor, designatedL-5-HT_(1F), was deposited on Dec. 27, 1991, and accorded ATCC AccessionNo. ATCC 10957. These preparations were suspended in 50 mM Tris-HClbuffer (pH 7.4 at 37° C.) containing 10 mM MgCl₂, 0.2 mM EDTA, 10 μMpargyline, and 0.1% ascorbate. The potency of α₁ antagonists wasdetermined in competition binding assay by incubation for 30 minutes at37° C. in the presence of 5 nM [3H]serotonin. Nonspecific binding wasdetermined in the presence of 10 μM serotonin. The bound radioligand wasseparated by filtration through GF/B filters using a cell harvester.

[0081] Human 5HT₂ Receptors: The coding sequence of the human 5HT₂receptor was obtained from a human brain cortex cDNA library, and clonedinto the cloning site of pCEXV-3 eukaryotic expression vector. Thisconstruct was transfected into COS-7 cells by the DEAE-dextran method.Cells were harvested after 72 hours and lysed by sonication in 5 mMTris-HCl, 5 mM EDTA, pH 7.5. This cell line was deposited with the ATCCon Oct. 31, 1989, designated as L-NGC-5HT₂, and was accorded ATCCAccession No. CRL 10287. The cell lysates were centrifuged at 1000 rpmfor 5 minutes at 4° C., and the supernatant was centrifuged at 30,000×gfor 20 minutes at 4° C. The pellet was suspended in 50 mM Tris-HClbuffer (pH 7.7 at room temperature) containing 10 mM MgSO₄, 0.5 mM EDTA,and 0.1% ascorbate. The potency of alpha-1 antagonists at 5HT2 receptorswas determined in equilibrium competition binding assays using[3H]ketanserin (1 nM). Nonspecific binding was defined by the additionof 10 μM mianserin. The bound radioligand was separated by filtrationthrough GF/B filters using a cell harvester.

[0082] Human Dopamine D2 Receptors: The potency of α₁ antagonists at theD2 receptor was determined using membrane preparations from COS-7 cellstransfected with the gene encoding the human D2 receptor. The codingregion for the human D2 receptor was obtained from a human striatum cDNAlibrary, and cloned into the cloning site of PCDNA 1 eukarioticexpression vector. The plasmid DNA for the D₂ receptor is designatedpcEXV-D2, and was deposited on Nov. 6, 1992 under ATCC Accession No. ATC75344. This construct was transfected into COS-7 cells by theDEAE-dextran method. Cells were harvested after 72 hours and lysed bysonication in 5 mM Tris-HCl, 5 mM EDTA, pH 7.5. The cell lysates werecentrifuged at 1000 rpm for 5 minutes at 4° C., and the supernatant wascentrifuged at 30,000×g for 20 minutes at 4° C. The pellet was suspendedin 50 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl₂,4 mM MgCl₂, and 0.1% ascorbic acid. The cell lysates were incubated with[3H]spiperone (2 nM), using 10 μM (+)Butaclamol to determine nonspecificbinding.

[0083] Other Dopamine receptors are prepared by known methods (D₃:Sokoloff, P. et al., Nature, 347, 146 (1990), and deposited with theEuropean Molecular Biological Laboratory (EMBL) Genbank as X53944; D₄:Van Tol, H. H. M., et al., Nature, 350, 610 (1991), and deposited withEMBL Genbank as X58497; D₅: Sunahara, R. K., et al., Nature, 350, 614(1991), and deposited with EMBL Genbank as X58454-HU HD 5DR).

[0084] Determination of the Activity of α₁ Antagonists at CalciumChannels

[0085] The potency of α₁ antagonists at calcium channels was determinedin competition binding assays of [3H]nitrendipine to membrane fragmentsof rat cardiac muscle, essentially as described by Glossman and Ferry(Methods in Enzymology 109:513-550, 1985). Briefly, the tissue wasminced and homogenized in 50 mM Tris-HCl (pH 7.4) containing 0.1 mMphenylmethylsulfonyl fluoride. The homogenates were centrifuged at 1000g for 15 minutes, the resulting supernatant was centrifuged at 45,000 gfor 15 minutes. The 45,000 g pellet was suspended in buffer andcentrifuged a second time. Aliquots of membrane protein were incubatedfor 30 minutes at 37° C. in the presence of [3H]nitrendipine (1 nM), andnonspecific binding was determined in the presence of 10 μM nifedipine.The bound radioligand was separated by filtration through GF/B filtersusing a cell harvester.

EXAMPLE 10 Functional Properties of α₁ Antagonists in the Human Prostate

[0086] The efficacy of α₁ adrenergic antagonists for the treatment ofbenign prostatic hyperplasia (BPH) is related to their ability to elicitrelaxation of prostate smooth muscle. An index of this efficacy can beobtained by determining the potency of α₁ antagonists to antagonize thecontraction of human prostatic tissue induced by an α₁ agonist “invitro”. Furthermore, by comparing the potency of subtype selective α₁antagonists in binding assays using human α₁ receptors with theirpotency to inhibit agonist-induced smooth muscle contraction, it ispossible to determine which of the α₁ adrenergic receptor subtypes isinvolved in the contraction of prostate smooth muscle.

[0087] Methods: Prostatic adenomas were obtained at the time of surgeryfrom patients with symptomatic BPH. These were cut into longitudinalstrips of 15 mm long and 2-4 mm wide, and suspended in 5 ml organ bathscontaining Krebs buffer (pH 7.4). The baths were maintained at 37° C.and continuously oxygenated with 5% CO₂ and 95% O₂. Isometric tensionwas measured with a Grass Instrument FT03 force transducer interfacedwith a computer. Tissue strips were contracted with varyingconcentrations of phenylephrine after incubating for 20 minutes in theabsence and presence of at least three different concentrations ofantagonist. Dose-response curves for phenylephrine were constructed, andthe antagonist potency (pA₂) was estimated by the dose-ratio method. Theconcentration of some antagonists in the tissue bath was assessed bymeasuring the displacement of [3H]prazosin by aliquots of the bathmedium, using membrane preparations of the cloned human α_(1C) receptor.This control was necessary to account for losses of antagonist due toadsorption to the tissue bath and/or metabolism during the time theantagonists were equilibrated with the prostate tissue.

[0088] Results:

[0089] Table 1 shows that the pA₂ values measured for a series of α₁antagonists in human prostate tissue correlate closely (r=0.76) with thecorresponding pK_(i) values measured in the α_(1C) receptor assays. Incontrast, the human prostate pA₂ values correlate poorly with the pK_(i)values measured at the α_(1A) (r=−0.06) and α_(1B) (r=−0.24) adrenergicreceptors. (See FIG. 2.) Thus, antagonists which are more potent atblocking the α_(1C) adrenergic receptor are more effective at blockingthe contraction of the human prostate than antagonists which are morepotent at the α_(1A) or α₁ adrenergic receptors. In addition,antagonists which are selective for the α_(1C) receptor will have abetter therapeutic ratio than nonselective α antagonists.

[0090] With SNAP 5036 (11), the low pA₂ observed in the prostate may beattributed to tissue absorption or metabolism.

[0091] Table 2 illustrates the cross reactivity of α₁ antagonists atother receptors such as α_(2A), α_(2B), α_(2C), histamine H₁, H₂,serotonin 5-HT_(1Dα), 5-HT_(1Dβ), 5-HT_(1E), 5-HT_(1F), 5-HT₂, anddopamine D₂. Only compounds SNAP 5036, 5041, and 5089 have bindingaffinities which are greater than ten-fold higher at α_(1C) receptorsthan the binding affinities at other receptors. TABLE 1 COMPARISON OFTHE BINDING POTENCY (pK₁) of ALPHA-1 ANTAGONISTS IN CLONED HUMANRECEPTORS AND THEIR PROTENCY (pA₂) TO INHIBIT PROSTATE SMOOTH MUSCLECONTRACTION Human Alpha-1 Adrenergic (pK₁) Human Compound a1A a1B a1CProstate (pA)  1 Prazosin 9.48 9.26 9.23 9.08  2 Compound 2 5.98 6.578.87 8.94  3 A-30360 7.49 7.86 8.52 8.72  4 5-Methyl-Urapidil 7.79 6.778.35 8.38  5 Indoramin 6.74 7.39 8.35 7.86  6 SKF-104856 8.48 7.50 7.607.66  7 Compound 7 6.82 7.18 8.42 7.63  8 Compound 8 6.52 7.07 8.48 7.46 9 Compound 9 6.12 6.76 7.83 7.41 10 Terazosin 8.46 8.71 8.16 7.30 11Compound 11 6.81 7.14 8.36 6.64

[0092] TABLE 2 CROSS REACTIVITY OF ALPHA-1 ANTAGONISTS AT CLONED HUMANRECETORS (pK,) Alpha-1 Adrenergic Alpha-2 Adrenergic Histamine SerotoninDopamine Calcium Compound a1A a1B a1C a2a a2b a2c H1 H2 5HT1Da 5HT1Db5HT1E 5HT1F 5HT2 D2 Channel Terazosin 8.46 8.71 8.16 6.26 7.51 6.64 4.005.04 <6.0 <6.0 <5.0 <5.0 <5.0 <5.0 5.19 Prazosin 9.48 9.26 9.23 6.767.64 7.65 4.00 5.19 <5.0 <5.0 ND ND <6.0 <5.0 4.57 5-Methylurapidil 7.796.77 8.35 6.63 7.38 6.88 5.16 4.47 7.30 6.82 ND ND <6.0 <5.0 NDIndoramin 6.74 7.39 8.35 4.94 5.72 5.22 7.37 5.63 <6.0 <6.0 <5.0 <5.0<7.0 <8.0 4.53 Compound 11 6.81 7.14 8.36 6.86 6.90 6.92 5.74 7.45 <6.0<6.0 <5.0 <5.0 <7.0 <6.0 5.18 A-30360 7.49 7.86 8.52 6.69 6.37 6.23 6.035.77 <6.0 <6.0 <5.0 <5.0 <8.0 <9.0 5.26 Compound 7 6.82 7.18 8.42 6.196.07 6.09 7.59 6.02 <6.0 <5.0 <5.0 <5.0 <6.0 <7.0 4.79 Compound 9 6.126.76 7.83 5.80 5.69 5.90 7.29 5.44 <6.0 <6.0 <5.0 <5.0 <7.0 <7.0 4.44SKF-104856 8.48 7.50 7.60 7.30 8.49 7.60 5.59 5.84 <7.0 <7.0 <6.0 <7.0<6.0 <7.0 4.68 S-Niguldipine 6.72 7.07 8.75 6.19 5.24 6.43 6.78 6.24 NDND ND ND <7.0 <7.0 8.04 Compound 8 6.52 7.07 8.48 5.99 6.12 5.77 6.676.11 <6.0 <5.0 <5.0 <5.0 <7.0 <6.0 6.87 Compound 2 5.98 6.57 8.87 5.485.93 5.88 7.16 7.48 <7.0 <6.0 <5.0 <5.0 <6.0 <7.0 6.13

EXAMPLE 11 Functional Properties of α₁ Antagonists on Rat OrthostaticHypertension

[0093] We have identified a large series of compounds (well over 150compounds, data not shown) which exemplify the hereinabove describedproperties of antagonists highly selective for the α_(1C) adrenergicreceptor. That is, these compounds are highly selective Alpha 1cantagonists which have less than 10 fold the affinity at cloned humanAlpha 1a, Alpha 1b, Alpha 2a, Alpha 2b, Alpha 2c, Histamine H1, DopamineD2 and Serotonin receptors. In addition, these compounds have 10 foldlower affinity at calcium channels (data not shown). We designated fiveof these highly selective antagonists for the α_(1C) adrenergic receptoras drugs 21-25 and used them to further characterize highly selectiveantagonists for the α_(1C) adrenergic receptor.

[0094] In addition, a number of these selective alpha 1c antagonists arepotent at inhibiting the phenylephrine stimulated contraction of humanprostate as described in Example 10. This is a well established protocolfor evaluation the efficacy of drugs which may be useful for thetreatment of BPH.

[0095] In addition, we have examined a number of selective alpha 1cantagonists in an in vivo canine prostate model (Felson, D., et al., J.Urol., 141, 1230-1233 (1989))which is a well characterized model forevaluating the efficacy of BPH drugs (data not shown). In this model,selective alpha 1c antagonists increase urethral pressure at doses whichdo not produce significant decreases in canine blood pressure. Incontrast, nonselective alpha 1 antagonists do not have as large aseparation between the effects on urethral pressure and the effects onblood pressure. These observations support our premise that a selectivealpha 1c antagonist will have a better safety profile than anonselective alpha 1 antagonist. We have further characterized selectivealpha 1c antagonists in a rat orthostatic hypotension model. This modelgives information on the vascular effects of drugs which may beindicative of their ability to produce dizziness in patients (Hieble, J.P., et al., Cardiovascular Pharmacology, 15, 845 (1990)). Our objectivewas to characterize the effects of selective alpha 1c antagonists on ratorthostatic hypotension and contrast the results with those obtainedusing nonselective alpha 1 antagonists.

[0096] Methods

[0097] Rat Orthostatic Hypotension Model

[0098] Adult male Sprague-Dawley normotensive rats were anesthetizedwith sodium pentobarbital (45 mg/kg, i.v.). The femoral vein and arteryof the right hindlimb were cannulated for drug administration and bloodpressure monitoring, respectively. Heart rate was determined by acardiotachometer triggered by the blood pressure pulse. The rats weresecured in the supine position to a board that could be tilted 90degrees. When blood pressure and heart rate had stabilized, the ratswere subjected to a 90 degree vertical (head up) tilt for 60 seconds.Changes in blood pressure and heart rate from pre-tilt levels weremonitored continuously. The rats were returned to the supine positionand blood pressure and heart rate were allowed to stabilize. Either anantagonist selective for the α_(1C) adrenergic receptor (designated drug21, 22, 23, 24 or 25), an antagonist nonselective for the α_(1C)adrenergic receptor (Prazosin or Terazosin) or saline was thenadministered through venous cannula, either as an i.v. bolus or as aninfusion. When blood pressure had stabilized, the rats were subjected toa second tilt and blood pressure and heart rate were recorded asdescribed above. Most saline treated rats typically exhibit a greaterability to return their blood pressure toward pre-tilt levels during thesecond tilt. Data from the second tilt are used in statistical analysis.

[0099] Results

[0100] Table 3 shows that while nonselective alpha 1 antagonists producesignificant effects on orthostatic hypotension, selective alpha 1cantagonists do not produce significant effects. More specifically,Prazosin and Terazosin consistently cause orthostasis at the lowest dose(10 ug/kg) and, in some rats, in a dose-dependent manner. Drug 21 causesorthostasis only at the highest dose (1000 ug/kg) in 2 out of 4 rats,while the other antagonists selective for the α_(1C) adrenergic receptorcaused no orthostasis at the highest dose. Placebo and 22, 23, 24, 25did not induce orthostasis at any dose. Taken all together, this is apositive result since it is believed that orthostatic hypotensioncontributes to the dizziness observed clinically with nonselective alpha1 antagonists. This further supports our premise that a selective alpha1c antagonist will have a better safety profile than a nonselectivealpha 1 antagonist. TABLE 3 Summary of Studies on Drug Effects onOrthostasis Dose 1 Dose 2 Dose 3 10 ug/kg 100 ug/kg 1000 ug/kgorthostatic BP orthostatic BP orthostatic BP Drug n fall in BP fall fallin BP fall fall in BP fall Notes Placebo (DMSO) 3 − − − − − − Prezosin4 + + ++ or +++ ++ ++ or +++ +++ Terazosin 2 + + ++ or +++ ++ ++ or ++++++ 21 4 − + − ++ +/− +++ (+ in 2/4) 22 3 − + − ++ − +++ 23 6 − − − −− + 24 6 − − − +/− − + 25 4 − − +/− − − − (+ in 1/4)

[0101]

1 6 2140 base pairs nucleic acid single unknown DNA (genomic) N N CDS178..1893 1 CCGGGCCAGG CACGTCCGCT CTCGGACAGC CGCTCCGCGT CACAGGAACTTGGGCAGGAC 60 CCGACGGGAC CCGTGCGCGG AGCTGCATCT GGAGCCCCGC GGCTATGCCCTGTGCTCCCC 120 TCCTGCCGGC CGCTCGTTCT GTGCCCCCGG CCCGGCCACC GACGGCCGCGCGTTGAG 177 ATG ACT TTC CGC GAT CTC CTG AGC GTC AGT TTC GAG GGA CCC CGCCCG 225 Met Thr Phe Arg Asp Leu Leu Ser Val Ser Phe Glu Gly Pro Arg Pro1 5 10 15 GAC AGC AGC GCA GGG GGC TCC AGC GCG GGC GGC GGC GGG GGC AGCGCG 273 Asp Ser Ser Ala Gly Gly Ser Ser Ala Gly Gly Gly Gly Gly Ser Ala20 25 30 GGC GGC GCG GCC CCC TCG GAG GGC CCG GCG GTG GGC GGC GTG CCG GGG321 Gly Gly Ala Ala Pro Ser Glu Gly Pro Ala Val Gly Gly Val Pro Gly 3540 45 GGC GCG GGC GGC GGC GGC GGC GTG GTG GGC GCA GGC AGC GGC GAG GAC369 Gly Ala Gly Gly Gly Gly Gly Val Val Gly Ala Gly Ser Gly Glu Asp 5055 60 AAC CGG AGC TCC GCG GGG GAG CCG GGG AGC GCG GGC GCG GGC GGC GAC417 Asn Arg Ser Ser Ala Gly Glu Pro Gly Ser Ala Gly Ala Gly Gly Asp 6570 75 80 GTG AAT GGC ACG GCG GCC GTC GGG GGA CTG GTG GTG AGC GCG CAG GGC465 Val Asn Gly Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gln Gly 8590 95 GTG GGC GTG GGC GTC TTC CTG GCA GCC TTC ATC CTT ATG GCC GTG GCA513 Val Gly Val Gly Val Phe Leu Ala Ala Phe Ile Leu Met Ala Val Ala 100105 110 GGT AAC CTG CTT GTC ATC CTC TCA GTG GCC TGC AAC CGC CAC CTG CAG561 Gly Asn Leu Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln 115120 125 ACC GTC ACC AAC TAT TTC ATC GTG AAC CTG GCC GTG GCC GAC CTG CTG609 Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu 130135 140 CTG AGC GCC ACC GTA CTG CCC TTC TCG GCC ACC ATG GAG GTT CTG GGC657 Leu Ser Ala Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly 145150 155 160 TTC TGG GCC TTT GGC CGC GCC TTC TGC GAC GTA TGG GCC GCC GTGGAC 705 Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp165 170 175 GTG CTG TGC TGC ACG GCC TCC ATC CTC AGC CTC TGC ACC ATC TCCGTG 753 Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Thr Ile Ser Val180 185 190 GAC CGG TAC GTG GGC GTG CGC CAC TCA CTC AAG TAC CCA GCC ATCATG 801 Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala Ile Met195 200 205 ACC GAG CGC AAG GCG GCC GCC ATC CTG GCC CTG CTC TGG GTC GTAGCC 849 Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu Leu Trp Val Val Ala210 215 220 CTG GTG GTG TCC GTA GGG CCC CTG CTG GGC TGG AAG GAG CCC GTGCCC 897 Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu Pro Val Pro225 230 235 240 CCT GAC GAG CGC TTC TGC GGT ATC ACC GAG GAG GCG GGC TACGCT GTC 945 Pro Asp Glu Arg Phe Cys Gly Ile Thr Glu Glu Ala Gly Tyr AlaVal 245 250 255 TTC TCC TCC GTG TGC TCC TTC TAC CTG CCC ATG GCG GTC ATCGTG GTC 993 Phe Ser Ser Val Cys Ser Phe Tyr Leu Pro Met Ala Val Ile ValVal 260 265 270 ATG TAC TGC CGC GTG TAC GTG GTC GCG CGC AGC ACC ACG CGCAGC CTC 1041 Met Tyr Cys Arg Val Tyr Val Val Ala Arg Ser Thr Thr Arg SerLeu 275 280 285 GAG GCA GGC GTC AAG CGC GAG CGA GGC AAG GCC TCC GAG GTGGTG CTG 1089 Glu Ala Gly Val Lys Arg Glu Arg Gly Lys Ala Ser Glu Val ValLeu 290 295 300 CGC ATC CAC TGT CGC GGC GCG GCC ACG GGC GCC GAC GGG GCGCAC GGC 1137 Arg Ile His Cys Arg Gly Ala Ala Thr Gly Ala Asp Gly Ala HisGly 305 310 315 320 ATG CGC AGC GCC AAG GGC CAC ACC TTC CGC AGC TCG CTCTCC GTG CGC 1185 Met Arg Ser Ala Lys Gly His Thr Phe Arg Ser Ser Leu SerVal Arg 325 330 335 CTG CTC AAG TTC TCC CGT GAG AAG AAA GCG GCC AAG ACTCTG GCC ATC 1233 Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr LeuAla Ile 340 345 350 GTC GTG GGT GTC TTC GTG CTC TGC TGG TTC CCT TTC TTCTTT GTC CTG 1281 Val Val Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe PheVal Leu 355 360 365 CCG CTC GGC TCC TTG TTC CCG CAG CTG AAG CCA TCG GAGGGC GTC TTC 1329 Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Ser Glu GlyVal Phe 370 375 380 AAG GTC ATC TTC TGG CTC GGC TAC TTC AAC AGC TGC GTGAAC CCG CTC 1377 Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val AsnPro Leu 385 390 395 400 ATC TAC CCC TGT TCC AGC CGC GAG TTC AAG CGC GCCTTC CTC CGT CTC 1425 Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala PheLeu Arg Leu 405 410 415 CTG CGC TGC CAG TGC CGT CGT CGC CGG CGC CGC CGCCCT CTC TGG CGT 1473 Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg ProLeu Trp Arg 420 425 430 GTC TAC GGC CAC CAC TGG CGG GCC TCC ACC AGC GGCCTG CGC CAG GAC 1521 Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly LeuArg Gln Asp 435 440 445 TGC GCC CCG AGT TCG GGC GAC GCG CCC CCC GGA GCGCCG CTG GCC CTC 1569 Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala ProLeu Ala Leu 450 455 460 ACC GCG CTC CCC GAC CCC GAC CCC GAA CCC CCA GGCACG CCC GAG ATG 1617 Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly ThrPro Glu Met 465 470 475 480 CAG GCT CCG GTC GCC AGC CGT CGA AAG CCA CCCAGC GCC TTC CGC GAG 1665 Gln Ala Pro Val Ala Ser Arg Arg Lys Pro Pro SerAla Phe Arg Glu 485 490 495 TGG AGG CTG CTG GGG CCG TTC CGG AGA CCC ACGACC CAG CTG CGC GCC 1713 Trp Arg Leu Leu Gly Pro Phe Arg Arg Pro Thr ThrGln Leu Arg Ala 500 505 510 AAA GTC TCC AGC CTG TCG CAC AAG ATC CGC GCCGGG GGC GCG CAG CGC 1761 Lys Val Ser Ser Leu Ser His Lys Ile Arg Ala GlyGly Ala Gln Arg 515 520 525 GCA GAG GCA GCG TGC GCC CAG CGC TCA GAG GTGGAG GCT GTG TCC CTA 1809 Ala Glu Ala Ala Cys Ala Gln Arg Ser Glu Val GluAla Val Ser Leu 530 535 540 GGC GTC CCA CAC GAG GTG GCC GAG GGC GCC ACCTGC CAG GCC TAC GAA 1857 Gly Val Pro His Glu Val Ala Glu Gly Ala Thr CysGln Ala Tyr Glu 545 550 555 560 TTG GCC GAC TAC AGC AAC CTA CGG GAG ACCGAT ATT TAAGGACCCC 1903 Leu Ala Asp Tyr Ser Asn Leu Arg Glu Thr Asp Ile565 570 AGAGCTAGGC CGCGGAGTGT GCTGGGCTTG GGGGTAAGGG GGACCAGAGAGGCGGGCTGG 1963 TGTTCTAAGA GCCCCCGTGC AAATCGGAGA CCCGGAAACT GATCAGGGCAGCTGCTCTGT 2023 GACATCCCTG AGGAACTGGG CAGAGCTTGA GGCTGGAGCC CTTGAAAGGTGAAAAGTAGT 2083 GGGGCCCCCT GCTGGACTCA GGTGCCCAGA ACTCTTTTCT TAGAAGGGAGAGGCTGC 2140 572 amino acids amino acid linear protein 2 Met Thr Phe ArgAsp Leu Leu Ser Val Ser Phe Glu Gly Pro Arg Pro 1 5 10 15 Asp Ser SerAla Gly Gly Ser Ser Ala Gly Gly Gly Gly Gly Ser Ala 20 25 30 Gly Gly AlaAla Pro Ser Glu Gly Pro Ala Val Gly Gly Val Pro Gly 35 40 45 Gly Ala GlyGly Gly Gly Gly Val Val Gly Ala Gly Ser Gly Glu Asp 50 55 60 Asn Arg SerSer Ala Gly Glu Pro Gly Ser Ala Gly Ala Gly Gly Asp 65 70 75 80 Val AsnGly Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gln Gly 85 90 95 Val GlyVal Gly Val Phe Leu Ala Ala Phe Ile Leu Met Ala Val Ala 100 105 110 GlyAsn Leu Leu Val Ile Leu Ser Val Ala Cys Asn Arg His Leu Gln 115 120 125Thr Val Thr Asn Tyr Phe Ile Val Asn Leu Ala Val Ala Asp Leu Leu 130 135140 Leu Ser Ala Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly 145150 155 160 Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala ValAsp 165 170 175 Val Leu Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Thr IleSer Val 180 185 190 Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr ProAla Ile Met 195 200 205 Thr Glu Arg Lys Ala Ala Ala Ile Leu Ala Leu LeuTrp Val Val Ala 210 215 220 Leu Val Val Ser Val Gly Pro Leu Leu Gly TrpLys Glu Pro Val Pro 225 230 235 240 Pro Asp Glu Arg Phe Cys Gly Ile ThrGlu Glu Ala Gly Tyr Ala Val 245 250 255 Phe Ser Ser Val Cys Ser Phe TyrLeu Pro Met Ala Val Ile Val Val 260 265 270 Met Tyr Cys Arg Val Tyr ValVal Ala Arg Ser Thr Thr Arg Ser Leu 275 280 285 Glu Ala Gly Val Lys ArgGlu Arg Gly Lys Ala Ser Glu Val Val Leu 290 295 300 Arg Ile His Cys ArgGly Ala Ala Thr Gly Ala Asp Gly Ala His Gly 305 310 315 320 Met Arg SerAla Lys Gly His Thr Phe Arg Ser Ser Leu Ser Val Arg 325 330 335 Leu LeuLys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Ala Ile 340 345 350 ValVal Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe Val Leu 355 360 365Pro Leu Gly Ser Leu Phe Pro Gln Leu Lys Pro Ser Glu Gly Val Phe 370 375380 Lys Val Ile Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu 385390 395 400 Ile Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu ArgLeu 405 410 415 Leu Arg Cys Gln Cys Arg Arg Arg Arg Arg Arg Arg Pro LeuTrp Arg 420 425 430 Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly LeuArg Gln Asp 435 440 445 Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly AlaPro Leu Ala Leu 450 455 460 Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro ProGly Thr Pro Glu Met 465 470 475 480 Gln Ala Pro Val Ala Ser Arg Arg LysPro Pro Ser Ala Phe Arg Glu 485 490 495 Trp Arg Leu Leu Gly Pro Phe ArgArg Pro Thr Thr Gln Leu Arg Ala 500 505 510 Lys Val Ser Ser Leu Ser HisLys Ile Arg Ala Gly Gly Ala Gln Arg 515 520 525 Ala Glu Ala Ala Cys AlaGln Arg Ser Glu Val Glu Ala Val Ser Leu 530 535 540 Gly Val Pro His GluVal Ala Glu Gly Ala Thr Cys Gln Ala Tyr Glu 545 550 555 560 Leu Ala AspTyr Ser Asn Leu Arg Glu Thr Asp Ile 565 570 1738 base pairs nucleic acidsingle unknown DNA (genomic) N N CDS 124..1683 3 GCCAGGAGGG CGCCTCTGGGAAGAAGACCA CGGGGGAAGC AAAGTTTCAG GGCAGCTGAG 60 GAGCCTTCGC CGCAGCCCTTCCGAGCCCAA TCATCCCCCA GGCTATGGAG GGCGGACTCT 120 AAG ATG AAT CCC GAC CTGGAC ACC GGC CAC AAC ACA TCA GCA CCT GCC 168 Met Asn Pro Asp Leu Asp ThrGly His Asn Thr Ser Ala Pro Ala 1 5 10 15 CAC TGG GGA GAG TTG AAA AATGCC AAC TTC ACT GGC CCC AAC CAG ACC 216 His Trp Gly Glu Leu Lys Asn AlaAsn Phe Thr Gly Pro Asn Gln Thr 20 25 30 TCG AGC AAC TCC ACA CTG CCC CAGCTG GAC ATC ACC AGG GCC ATC TCT 264 Ser Ser Asn Ser Thr Leu Pro Gln LeuAsp Ile Thr Arg Ala Ile Ser 35 40 45 GTG GGC CTG GTG CTG GGC GCC TTC ATCCTC TTT GCC ATC GTG GGC AAC 312 Val Gly Leu Val Leu Gly Ala Phe Ile LeuPhe Ala Ile Val Gly Asn 50 55 60 ATC CTA GTC ATC TTG TCT GTG GCC TGC AACCGG CAC CTG CGG ACG CCC 360 Ile Leu Val Ile Leu Ser Val Ala Cys Asn ArgHis Leu Arg Thr Pro 65 70 75 ACC AAC TAC TTC ATT GTC AAC CTG GCC ATG GCCGAC CTG CTG TTG AGC 408 Thr Asn Tyr Phe Ile Val Asn Leu Ala Met Ala AspLeu Leu Leu Ser 80 85 90 95 TTC ACC GTC CTG CCC TTC TCA GCG GCC CTA GAGGTG CTC GGC TAC TGG 456 Phe Thr Val Leu Pro Phe Ser Ala Ala Leu Glu ValLeu Gly Tyr Trp 100 105 110 GTG CTG GGG CGG ATC TTC TGT GAC ATC TGG GCAGCC GTG GAT GTC CTG 504 Val Leu Gly Arg Ile Phe Cys Asp Ile Trp Ala AlaVal Asp Val Leu 115 120 125 TGC TGC ACA GCG TCC ATT CTG AGC CTG TGC GCCATC TCC ATC GAT CGC 552 Cys Cys Thr Ala Ser Ile Leu Ser Leu Cys Ala IleSer Ile Asp Arg 130 135 140 TAC ATC GGG GTG CGC TAC TCT CTG CAG TAT CCCACG CTG GTC ACC CGG 600 Tyr Ile Gly Val Arg Tyr Ser Leu Gln Tyr Pro ThrLeu Val Thr Arg 145 150 155 AGG AAG GCC ATC TTG GCG CTG CTC AGT GTC TGGGTC TTG TCC ACC GTC 648 Arg Lys Ala Ile Leu Ala Leu Leu Ser Val Trp ValLeu Ser Thr Val 160 165 170 175 ATC TCC ATC GGG CCT CTC CTT GGG TGG AAGGAG CCG GCA CCC AAC GAT 696 Ile Ser Ile Gly Pro Leu Leu Gly Trp Lys GluPro Ala Pro Asn Asp 180 185 190 GAC AAG GAG TGC GGG GTC ACC GAA GAA CCCTTC TAT GCC CTC TTC TCC 744 Asp Lys Glu Cys Gly Val Thr Glu Glu Pro PheTyr Ala Leu Phe Ser 195 200 205 TCT CTG GGC TCC TTC TAC ATC CCT CTG GCGGTC ATT CTA GTC ATG TAC 792 Ser Leu Gly Ser Phe Tyr Ile Pro Leu Ala ValIle Leu Val Met Tyr 210 215 220 TGC CGT GTC TAT ATA GTG GCC AAG AGA ACCACC AAG AAC CTA GAG GCA 840 Cys Arg Val Tyr Ile Val Ala Lys Arg Thr ThrLys Asn Leu Glu Ala 225 230 235 GGA GTC ATG AAG GAG ATG TCC AAC TCC AAGGAG CTG ACC CTG AGG ATC 888 Gly Val Met Lys Glu Met Ser Asn Ser Lys GluLeu Thr Leu Arg Ile 240 245 250 255 CAT TCC AAG AAC TTT CAC GAG GAC ACCCTT AGC AGT ACC AAG GCC AAG 936 His Ser Lys Asn Phe His Glu Asp Thr LeuSer Ser Thr Lys Ala Lys 260 265 270 GGC CAC AAC CCC AGG AGT TCC ATA GCTGTC AAA CTT TTT AAG TTC TCC 984 Gly His Asn Pro Arg Ser Ser Ile Ala ValLys Leu Phe Lys Phe Ser 275 280 285 AGG GAA AAG AAA GCA GCT AAG ACG TTGGGC ATT GTG GTC GGT ATG TTC 1032 Arg Glu Lys Lys Ala Ala Lys Thr Leu GlyIle Val Val Gly Met Phe 290 295 300 ATC TTG TGC TGG CTA CCC TTC TTC ATCGCT CTA CCG CTT GGC TCC TTG 1080 Ile Leu Cys Trp Leu Pro Phe Phe Ile AlaLeu Pro Leu Gly Ser Leu 305 310 315 TTC TCC ACC CTG AAG CCC CCC GAC GCCGTG TTC AAG GTG GTG TTC TGG 1128 Phe Ser Thr Leu Lys Pro Pro Asp Ala ValPhe Lys Val Val Phe Trp 320 325 330 335 CTG GGC TAC TTC AAC AGC TGC CTCAAC CCC ATC ATC TAC CCA TGC TCC 1176 Leu Gly Tyr Phe Asn Ser Cys Leu AsnPro Ile Ile Tyr Pro Cys Ser 340 345 350 AGC AAG GAG TTC AAG CGC GCT TTCGTG CGC ATC CTC GGG TGC CAG TGC 1224 Ser Lys Glu Phe Lys Arg Ala Phe ValArg Ile Leu Gly Cys Gln Cys 355 360 365 CGC GGC CGC GGC CGC CGC CGA CGCCGC CGC CGC CGT CGC CTG GGC GGC 1272 Arg Gly Arg Gly Arg Arg Arg Arg ArgArg Arg Arg Arg Leu Gly Gly 370 375 380 TGC GCC TAC ACC TAC CGG CCG TGGACG CGC GGC GGC TCG CTG GAG CGC 1320 Cys Ala Tyr Thr Tyr Arg Pro Trp ThrArg Gly Gly Ser Leu Glu Arg 385 390 395 TCG CAG TCG CGC AAG GAC TCG CTGGAC GAC AGC GGC AGC TGC CTG AGC 1368 Ser Gln Ser Arg Lys Asp Ser Leu AspAsp Ser Gly Ser Cys Leu Ser 400 405 410 415 GGC AGC CAG CGG ACC CTG CCCTCG GCC TCG CCG AGC CCG GGC TAC CTG 1416 Gly Ser Gln Arg Thr Leu Pro SerAla Ser Pro Ser Pro Gly Tyr Leu 420 425 430 GGC CGC GGC GCG CCA CCG CCAGTC GAG CTG TGC GCC TTC CCC GAG TGG 1464 Gly Arg Gly Ala Pro Pro Pro ValGlu Leu Cys Ala Phe Pro Glu Trp 435 440 445 AAG GCG CCC GGC GCC CTC CTGAGC CTG CCC GCG CCT GAG CCC CCC GGC 1512 Lys Ala Pro Gly Ala Leu Leu SerLeu Pro Ala Pro Glu Pro Pro Gly 450 455 460 CGC CGC GGC CGC CAC GAC TCGGGC CCG CTC TTC ACC TTC AAG CTC CTG 1560 Arg Arg Gly Arg His Asp Ser GlyPro Leu Phe Thr Phe Lys Leu Leu 465 470 475 ACC GAG CCC GAG AGC CCC GGGACC GAC GGC GGC GCC AGC AAC GGA GGC 1608 Thr Glu Pro Glu Ser Pro Gly ThrAsp Gly Gly Ala Ser Asn Gly Gly 480 485 490 495 TGC GAG GCC GCG GCC GACGTG GCC AAC GGG CAG CCG GGC TTC AAA AGC 1656 Cys Glu Ala Ala Ala Asp ValAla Asn Gly Gln Pro Gly Phe Lys Ser 500 505 510 AAC ATG CCC CTG GCG CCCGGG CAG TTT TAGGGCCCCC GTGCGCAGCT 1703 Asn Met Pro Leu Ala Pro Gly GlnPhe 515 520 TTCTTTCCCT GGGGAGGAAA ACATCGTGGG GGGGA 1738 520 amino acidsamino acid linear protein 4 Met Asn Pro Asp Leu Asp Thr Gly His Asn ThrSer Ala Pro Ala His 1 5 10 15 Trp Gly Glu Leu Lys Asn Ala Asn Phe ThrGly Pro Asn Gln Thr Ser 20 25 30 Ser Asn Ser Thr Leu Pro Gln Leu Asp IleThr Arg Ala Ile Ser Val 35 40 45 Gly Leu Val Leu Gly Ala Phe Ile Leu PheAla Ile Val Gly Asn Ile 50 55 60 Leu Val Ile Leu Ser Val Ala Cys Asn ArgHis Leu Arg Thr Pro Thr 65 70 75 80 Asn Tyr Phe Ile Val Asn Leu Ala MetAla Asp Leu Leu Leu Ser Phe 85 90 95 Thr Val Leu Pro Phe Ser Ala Ala LeuGlu Val Leu Gly Tyr Trp Val 100 105 110 Leu Gly Arg Ile Phe Cys Asp IleTrp Ala Ala Val Asp Val Leu Cys 115 120 125 Cys Thr Ala Ser Ile Leu SerLeu Cys Ala Ile Ser Ile Asp Arg Tyr 130 135 140 Ile Gly Val Arg Tyr SerLeu Gln Tyr Pro Thr Leu Val Thr Arg Arg 145 150 155 160 Lys Ala Ile LeuAla Leu Leu Ser Val Trp Val Leu Ser Thr Val Ile 165 170 175 Ser Ile GlyPro Leu Leu Gly Trp Lys Glu Pro Ala Pro Asn Asp Asp 180 185 190 Lys GluCys Gly Val Thr Glu Glu Pro Phe Tyr Ala Leu Phe Ser Ser 195 200 205 LeuGly Ser Phe Tyr Ile Pro Leu Ala Val Ile Leu Val Met Tyr Cys 210 215 220Arg Val Tyr Ile Val Ala Lys Arg Thr Thr Lys Asn Leu Glu Ala Gly 225 230235 240 Val Met Lys Glu Met Ser Asn Ser Lys Glu Leu Thr Leu Arg Ile His245 250 255 Ser Lys Asn Phe His Glu Asp Thr Leu Ser Ser Thr Lys Ala LysGly 260 265 270 His Asn Pro Arg Ser Ser Ile Ala Val Lys Leu Phe Lys PheSer Arg 275 280 285 Glu Lys Lys Ala Ala Lys Thr Leu Gly Ile Val Val GlyMet Phe Ile 290 295 300 Leu Cys Trp Leu Pro Phe Phe Ile Ala Leu Pro LeuGly Ser Leu Phe 305 310 315 320 Ser Thr Leu Lys Pro Pro Asp Ala Val PheLys Val Val Phe Trp Leu 325 330 335 Gly Tyr Phe Asn Ser Cys Leu Asn ProIle Ile Tyr Pro Cys Ser Ser 340 345 350 Lys Glu Phe Lys Arg Ala Phe ValArg Ile Leu Gly Cys Gln Cys Arg 355 360 365 Gly Arg Gly Arg Arg Arg ArgArg Arg Arg Arg Arg Leu Gly Gly Cys 370 375 380 Ala Tyr Thr Tyr Arg ProTrp Thr Arg Gly Gly Ser Leu Glu Arg Ser 385 390 395 400 Gln Ser Arg LysAsp Ser Leu Asp Asp Ser Gly Ser Cys Leu Ser Gly 405 410 415 Ser Gln ArgThr Leu Pro Ser Ala Ser Pro Ser Pro Gly Tyr Leu Gly 420 425 430 Arg GlyAla Pro Pro Pro Val Glu Leu Cys Ala Phe Pro Glu Trp Lys 435 440 445 AlaPro Gly Ala Leu Leu Ser Leu Pro Ala Pro Glu Pro Pro Gly Arg 450 455 460Arg Gly Arg His Asp Ser Gly Pro Leu Phe Thr Phe Lys Leu Leu Thr 465 470475 480 Glu Pro Glu Ser Pro Gly Thr Asp Gly Gly Ala Ser Asn Gly Gly Cys485 490 495 Glu Ala Ala Ala Asp Val Ala Asn Gly Gln Pro Gly Phe Lys SerAsn 500 505 510 Met Pro Leu Ala Pro Gly Gln Phe 515 520 1639 base pairsnucleic acid single unknown DNA (genomic) N N CDS 126..1523 5 CCAGCCAAACCACTGGCAGG CTCCCTCCAG CCGAGACCTT TTATTCCCGG CTCCCGAGCT 60 CCGCCTCCGCGCCAGCCCGG GAGGTGGCCC TGACAGCCGG ACCTCGCCCG GCCCCGGCTG 120 GGACC ATG GTGTTT CTC TCG GGA AAT GCT TCC GAC AGC TCC AAC TGC 167 Met Val Phe Leu SerGly Asn Ala Ser Asp Ser Ser Asn Cys 1 5 10 ACC CAA CCG CCG GCA CCG GTGAAC ATT TCC AAG GCC ATT CTG CTC GGG 215 Thr Gln Pro Pro Ala Pro Val AsnIle Ser Lys Ala Ile Leu Leu Gly 15 20 25 30 GTG ATC TTG GGG GGC CTC ATTCTT TTC GGG GTG CTG GGT AAC ATC CTA 263 Val Ile Leu Gly Gly Leu Ile LeuPhe Gly Val Leu Gly Asn Ile Leu 35 40 45 GTG ATC CTC TCC GTA GCC TGT CACCGA CAC CTG CAC TCA GTC ACG CAC 311 Val Ile Leu Ser Val Ala Cys His ArgHis Leu His Ser Val Thr His 50 55 60 TAC TAC ATC GTC AAC CTG GCG GTG GCCGAC CTC CTG CTC ACC TCC ACG 359 Tyr Tyr Ile Val Asn Leu Ala Val Ala AspLeu Leu Leu Thr Ser Thr 65 70 75 GTG CTG CCC TTC TCC GCC ATC TTC GAG GTCCTA GGC TAC TGG GCC TTC 407 Val Leu Pro Phe Ser Ala Ile Phe Glu Val LeuGly Tyr Trp Ala Phe 80 85 90 GGC AGG GTC TTC TGC AAC ATC TGG GCG GCA GTGGAT GTG CTG TGC TGC 455 Gly Arg Val Phe Cys Asn Ile Trp Ala Ala Val AspVal Leu Cys Cys 95 100 105 110 ACC GCG TCC ATC ATG GGC CTC TGC ATC ATCTCC ATC GAC CGC TAC ATC 503 Thr Ala Ser Ile Met Gly Leu Cys Ile Ile SerIle Asp Arg Tyr Ile 115 120 125 GGC GTG AGC TAC CCG CTG CGC TAC CCA ACCATC GTC ACC CAG AGG AGG 551 Gly Val Ser Tyr Pro Leu Arg Tyr Pro Thr IleVal Thr Gln Arg Arg 130 135 140 GGT CTC ATG GCT CTG CTC TGC GTC TGG GCACTC TCC CTG GTC ATA TCC 599 Gly Leu Met Ala Leu Leu Cys Val Trp Ala LeuSer Leu Val Ile Ser 145 150 155 ATT GGA CCC CTG TTC GGC TGG AGG CAG CCGGCC CCC GAG GAC GAG ACC 647 Ile Gly Pro Leu Phe Gly Trp Arg Gln Pro AlaPro Glu Asp Glu Thr 160 165 170 ATC TGC CAG ATC AAC GAG GAG CCG GGC TACGTG CTC TTC TCA GCG CTG 695 Ile Cys Gln Ile Asn Glu Glu Pro Gly Tyr ValLeu Phe Ser Ala Leu 175 180 185 190 GGC TCC TTC TAC CTG CCT CTG GCC ATCATC CTG GTC ATG TAC TGC CGC 743 Gly Ser Phe Tyr Leu Pro Leu Ala Ile IleLeu Val Met Tyr Cys Arg 195 200 205 GTC TAC GTG GTG GCC AAG AGG GAG AGCCGG GGC CTC AAG TCT GGC CTC 791 Val Tyr Val Val Ala Lys Arg Glu Ser ArgGly Leu Lys Ser Gly Leu 210 215 220 AAG ACC GAC AAG TCG GAC TCG GAG CAAGTG ACG CTC CGC ATC CAT CGG 839 Lys Thr Asp Lys Ser Asp Ser Glu Gln ValThr Leu Arg Ile His Arg 225 230 235 AAA AAC GCC CCG GCA GGA GGC AGC GGGATG GCC AGC GCC AAG ACC AAG 887 Lys Asn Ala Pro Ala Gly Gly Ser Gly MetAla Ser Ala Lys Thr Lys 240 245 250 ACG CAC TTC TCA GTG AGG CTC CTC AAGTTC TCC CGG GAG AAG AAA GCG 935 Thr His Phe Ser Val Arg Leu Leu Lys PheSer Arg Glu Lys Lys Ala 255 260 265 270 GCC AAA ACG CTG GGC ATC GTG GTCGGC TGC TTC GTC CTC TGC TGG CTG 983 Ala Lys Thr Leu Gly Ile Val Val GlyCys Phe Val Leu Cys Trp Leu 275 280 285 CCT TTT TTC TTA GTC ATG CCC ATTGGG TCT TTC TTC CCT GAT TTC AAG 1031 Pro Phe Phe Leu Val Met Pro Ile GlySer Phe Phe Pro Asp Phe Lys 290 295 300 CCC TCT GAA ACA GTT TTT AAA ATAGTA TTT TGG CTC GGA TAT CTA AAC 1079 Pro Ser Glu Thr Val Phe Lys Ile ValPhe Trp Leu Gly Tyr Leu Asn 305 310 315 AGC TGC ATC AAC CCC ATC ATA TACCCA TGC TCC AGC CAA GAG TTC AAA 1127 Ser Cys Ile Asn Pro Ile Ile Tyr ProCys Ser Ser Gln Glu Phe Lys 320 325 330 AAG GCC TTT CAG AAT GTC TTG AGAATC CAG TGT CTC TGC AGA AAG CAG 1175 Lys Ala Phe Gln Asn Val Leu Arg IleGln Cys Leu Cys Arg Lys Gln 335 340 345 350 TCT TCC AAA CAT GCC CTG GGCTAC ACC CTG CAC CCG CCC AGC CAG GCC 1223 Ser Ser Lys His Ala Leu Gly TyrThr Leu His Pro Pro Ser Gln Ala 355 360 365 GTG GAA GGG CAA CAC AAG GACATG GTG CGC ATC CCC GTG GGA TCA AGA 1271 Val Glu Gly Gln His Lys Asp MetVal Arg Ile Pro Val Gly Ser Arg 370 375 380 GAG ACC TTC TAC AGG ATC TCCAAG ACG GAT GGC GTT TGT GAA TGG AAA 1319 Glu Thr Phe Tyr Arg Ile Ser LysThr Asp Gly Val Cys Glu Trp Lys 385 390 395 TTT TTC TCT TCC ATG CCC CGTGGA TCT GCC AGG ATT ACA GTG TCC AAA 1367 Phe Phe Ser Ser Met Pro Arg GlySer Ala Arg Ile Thr Val Ser Lys 400 405 410 GAC CAA TCC TCC TGT ACC ACAGCC CGG GTG AGA AGT AAA AGC TTT TTG 1415 Asp Gln Ser Ser Cys Thr Thr AlaArg Val Arg Ser Lys Ser Phe Leu 415 420 425 430 CAG GTC TGC TGC TGT GTAGGG CCC TCA ACC CCC AGC CTT GAC AAG AAC 1463 Gln Val Cys Cys Cys Val GlyPro Ser Thr Pro Ser Leu Asp Lys Asn 435 440 445 CAT CAA GTT CCA ACC ATTAAG GTC CAC ACC ATC TCC CTC AGT GAG AAC 1511 His Gln Val Pro Thr Ile LysVal His Thr Ile Ser Leu Ser Glu Asn 450 455 460 GGG GAG GAA GTCTAGGACAGGA AAGATGCAGA GGAAAGGGGA ATATCTTAGG 1563 Gly Glu Glu Val 465TACCATACCC TGGAGTTCTA GAGGATTCCT CGACAAGCTT ATTCCGATCC AGACATGATA 1623GATACATTGA TGAGTT 1639 466 amino acids amino acid linear protein 6 MetVal Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gln 1 5 10 15Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile Leu Leu Gly Val Ile 20 25 30Leu Gly Gly Leu Ile Leu Phe Gly Val Leu Gly Asn Ile Leu Val Ile 35 40 45Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60Ile Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 7580 Pro Phe Ser Ala Ile Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 9095 Val Phe Cys Asn Ile Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100105 110 Ser Ile Met Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val115 120 125 Ser Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg GlyLeu 130 135 140 Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile SerIle Gly 145 150 155 160 Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu AspGlu Thr Ile Cys 165 170 175 Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu PheSer Ala Leu Gly Ser 180 185 190 Phe Tyr Leu Pro Leu Ala Ile Ile Leu ValMet Tyr Cys Arg Val Tyr 195 200 205 Val Val Ala Lys Arg Glu Ser Arg GlyLeu Lys Ser Gly Leu Lys Thr 210 215 220 Asp Lys Ser Asp Ser Glu Gln ValThr Leu Arg Ile His Arg Lys Asn 225 230 235 240 Ala Pro Ala Gly Gly SerGly Met Ala Ser Ala Lys Thr Lys Thr His 245 250 255 Phe Ser Val Arg LeuLeu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270 Thr Leu Gly IleVal Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 280 285 Phe Leu ValMet Pro Ile Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290 295 300 Glu ThrVal Phe Lys Ile Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320Ile Asn Pro Ile Ile Tyr Pro Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330335 Phe Gln Asn Val Leu Arg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser 340345 350 Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu355 360 365 Gly Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg GluThr 370 375 380 Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp LysPhe Phe 385 390 395 400 Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr ValSer Lys Asp Gln 405 410 415 Ser Ser Cys Thr Thr Ala Arg Val Arg Ser LysSer Phe Leu Gln Val 420 425 430 Cys Cys Cys Val Gly Pro Ser Thr Pro SerLeu Asp Lys Asn His Gln 435 440 445 Val Pro Thr Ile Lys Val His Thr IleSer Leu Ser Glu Asn Gly Glu 450 455 460 Glu Val 465

What is claimed is:
 1. A method of treating benign prostatic hyperplasiain a subject which comprises administering to the subject atherapeutically effective amount of a compound which: a. binds to ahuman α_(1C) adrenergic receptor with a binding affinity greater thanten-fold higher than the binding affinity with which the compound bindsto a human α_(1A) adrenergic receptor, a human α_(1B) adrenergicreceptor, and a human histamine H₁ receptor; and b. binds to a human α₂adrenergic receptor with a binding affinity which is greater thanten-fold lower than the binding affinity with which the compound bindsto such α_(1C) adrenergic receptor.
 2. A method of claim 1, wherein thecompound additionally binds to a calcium channel with a binding affinitywhich is greater than ten-fold lower than the binding affinity withwhich the compound binds to the α_(1C) adrenergic receptor.
 3. A methodof claim 1 or 2, wherein the compound additionally binds to a dopamineD₂ receptor with a binding affinity which is greater than ten-fold lowerthan the binding affinity with which the compound binds to the α_(1C)adrenergic receptor.
 4. A method of claim 1, 2, or 3, wherein thecompound additionally binds to any serotonin receptor with a bindingaffinity which is greater than ten-fold lower than the binding affinitywith which the compound binds to the α_(1C) adrenergic receptor.
 5. Amethod of claim 1, 2, 3, or 4, wherein the compound additionally bindsto a dopamine D₃, D₄, or D₅ receptor with a binding affinity which isgreater than ten-fold lower than the binding affinity with which thecompound binds to the α_(1C) adrenergic receptor.
 6. A method of claim1, 2, 3, 4, or 5 wherein the compound additionally does not causeorthostatic fall in blood pressure at dosages effective to alleviatebenign prostatic hyperplasia.
 7. The method of claim 6 wherein thecompound additionally does not cause orthostatic fall in blood pressurein rats at a dosage of 10 micrograms of compound per kilogram of rat. 8.A method of claim 1, wherein the compound has the structure:


9. A method of claim 1, wherein the compound has the structure:


10. A method of claim 1, wherein the compound has the structure:


11. A method of claim 1, wherein the compound has the structure:


12. A method of inhibiting contraction of prostate tissue whichcomprises contacting the prostate tissue with an effectivecontraction-inhibiting amount of a compound which: a. binds to a humanα_(1C) adrenergic receptor with a binding affinity greater than ten-foldhigher than the binding affinity with which the compound binds to ahuman α_(1A) adrenergic receptor, a human α_(1B) adrenergic receptor,and a human histamine H₁ receptor; and b. binds to a human α₂ adrenergicreceptor with a binding affinity which is greater than ten-fold lowerthan the binding affinity with which the compound binds to such α_(1C)adrenergic receptor.